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Natur hat weder Kern 

Noch Schale, 

Alles ist sie mil einemmale — goethe 


J nilaclelpnia 

Copyright, 1939, by P. Blakiston's Son & Co., Inc. 


To the memory of 
<Jtfy ^Mother 

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merely to set forth my views on some biological problems 
which I had studied during a period of more than twenty- 
five years of research on the eggs of marine animals. I had 
thought my task easy; that it would be a simple matter to 
present my ideas and findings against the clearly delineated 
back-drop of general biological theories and on the basis of 
well-established fact. Merely to hint at the theories and 
to indicate briefly the factual evidence would suffice, I 
thought, to make myself clear to my colleagues. But in 
thinking thus, I had deluded myself; soon I realized that 
the back-drop had to be cleared in order to show its con- 
tours; that instead of a substantial platform I had only 
pieces of material that had first -to be ordered and put 
together before they could serve me. This work had to be 
done, I found, before I had a stage on which I could present 
my own ideas and let them speak their parts in my inter- 
pretation of the drama of life unfolding before our eyes. 
It thus became imperative for me to examine and to 
appraise hypothesis and factual evidence and to define first 
every problem the discussion of which I had projected for 
my book. 

Thus my book developed from a mere statement of my 
views for biologists engaged in a narrowly restricted field of 
investigation into a thoroughgoing presentation and dis- 
cussion of biological problems for a wider audience. To all 
those who look with interest upon the manifestations of life 
in animals and in man, who desire to know more, and more 



exactly what answers to their questions concerning life 
biology can give, this book would speak. Biologists in 
other fields, students in biology and related sciences and all 
who have general interest, I have endeavored to address in 
the following pages. 

I have the strong feeling that many thoughtful and 
serious men in engineering, practical physics and chemistry 
and laymen outside of these professions do not know what 
modern biology is; even medical men often wonder what 
this biology of to-day offers. They have genuine interest 
in animal life and give much reflection to biological ques- 
tions; yet present day biology leaves them untouched. For 
such readers text-books offer little. What the professional 
biologist denominates as biology — taxonomy, palaeontol- 
ogy, ecology, physiology, morphology, embryology, phyto- 
and zoo-geography, as well as genetics, biometry and the 
like, with their various subdivisions, each of which is again 
subdivided, as, for instance, cytology with its separate 
domains ruled by karyologists, " protoplasmatikers," 
''Golgiologists" and "mitochondriacs" etc., etc., — needs to 
be brought into relation with the world outside biological 
institutes and recording offices. To the uninitiated this 
biological regimentation inspires awe because it connotes 
the abstruse too far removed from everyday life. 

Even the most abstract truth needs to be expressed 
with simplicity and clearness and thus relate itself to 
everyday human experience. Complexity of expression is 
often a sign of incomplete knowledge and certainly it is 
not a sine qua non of learning, though there be those who 
consider profound and erudite that which they can never 
understand. However cloistered biology may be as a 
scientific research, as the science of life and having appeal 
to all men it should make itself articulate beyond its 
cloistered walls. I have endeavored in writing this book to 
express myself with such clearness that even the uninitiated 



can follow my argument at least in the main. At the same 
time I bring the reader at once into the arena of conflicting 
biological thought, for only thus, I think, can he realize the 
status of the science to-day. I trust that what to biologists 
are well-known facts I have presented in a manner that 
elicits their interest. 

Together with a definition of the general problems, my 
own work as well as my ideas are presented. The out- 
come of these is my theory which I set forth in a more 
explicit manner than heretofore. 

The conception upon which the book is built, though 
latent in my earlier researches, did not come fully awake 
until 1930 while I was enjoying the hospitality of the 
Kaiser- Wilhelm-Insti tut fur Biologie at Berlin-Dahlem. 
There I fell under the inspiration of Adolf von Harnack's 
personality. I like to feel that my work was influenced by 
the rich experience of personal contact with him. 

The studies which gave rise to my conception were made 
during some twenty years, largely at the Marine Biological 
Laboratory, Woods Hole, Mass. ; some few were made at the 
Zoological Station at Naples. For the support of many of 
these researches I am indebted to the late Mr. Julius 
Rosenwald, whose friendship I esteemed. However, this 
book could not have been finished but for the spontaneous 
and sympathetic understanding of my work shown by Dr. 
F. W. Keppel, President of the Carnegie Corporation. A 
grant from this corporation made possible a year's study 
necessary to complete the work. In the task of writing a 
book understandable also to non-biologists, I have further 
been sustained and encouraged by many friends, biologists, 
medical men, and others outside of these fields. 

The book may be divided into three parts. Part I, com- 
prising Introduction, Life and Experiment, Protoplasmic 
System, Ectoplasm, General Properties of the Ectoplasm 
and Water, though dealing primarily with the animal egg, 



embodies principles which concern the fundamental organi- 
zation of any living thing. Part II, including the Fertiliza- 
tion-process, the Fertilization-reaction, Parthenogenesis, 
Cell-division and Cleavage and Differentiation, discusses 
in particular the problems that refer directly to animal eggs 
in their earliest stages of development. Part III, embrac- 
ing chapters on the Chromosomes and Ectoplasm, Ecto- 
plasm and Evolution, and Conclusion, has to do with more 
or less theoretical discussions. Throughout the whole 
treatment, the principle that the cell is the biological unit is 
kept in mind. In particular, structure and function of the 
ectoplasm are emphasized; upon these my theory of the 
state of being alive is in large measure grounded. 

Concerning the literature cited, I should point out that 
I have made no attempt to refer to all of the many original 
papers which I have studied and abstracted. Instead, with 
each revision of the manuscript I have reduced the number 
of titles in the bibliography. Nevertheless, the list of titles 
retained is ample for the argument set forth. 

E. E. J. 

Paris, I* raxce, 
November, 1938. 





i . Introduction ^ 

2. Life and Experiaient i0 

3. The Protoplasmic System 3 1 

4. The Ectoplasm 75 

5. General Properties of the Ectoplasm 104 

6. Water i2 4 

7. The Fertilization-process i 47 

8. The Fertilization-reaction t ^4 

9. Parthenogenesis 20 ® 
10. Cell-division 2 47 
1 1 . Cleavage and Differentiation 280 

12. Chromosomes and Ectoplasm 34° 

13. Ectoplasm and Evolution 354 

14. Conclusion 3® 2 

15. Bibliography 37° 
Index of Authors 3^ >i 
Index of Subjects 3°4 



The realm of living things being a part of nature 
is contiguous to the non-living world. Living things have 
material composition, are made up finally of units, mole- 
cules, atoms, and electrons, as surely as any non-living 
matter. Like all forms in nature they have chemical struc- 
ture and physical properties, are physico-chemical systems. 
As such they obey the laws of physics and chemistry. 
Would one deny this fact, one would thereby deny the 
possibility of any scientific investigation of living things. 
No matter what beliefs we entertain, the noblest and purest, 
concerning life as something apart from physical and chemi- 
cal phenomena, we can not with the mental equipment 
which we now possess reach any estimate of living things as 
apart from the remainder of the physico-chemical world. 

But although any living thing, being matter, is a physico- 
chemical system, it differs from matter which constitutes 
the non-living. This difference exists and would continue 
to exist were some chemist at this moment to succeed to 
synthesize out of non-living matter a living thing. The 
analysis of living things reveals that they are composed of 
no peculiar chemical elements — instead, they are made up 
of those most commonly occurring. The difference can not 
then be attributed to the elements. To be sure, certain 
complex compounds, as proteins, carbohydrates and lipins 
(fats and fat-like substances) — themselves compounded 
mainly of the commonly occurring elements, carbon, hydro- 
gen and oxygen, and never of rare elements — are peculiar 



to living matter; but the synthesis of protein-like bodies, 
of sugar and of fat as well as the synthesis of thyroxin 
(a compound in the internal secretion of the thyroid gland), 
of products of other internal secretions and of vitamins must 
dissipate whatever belief may have lingered on since 
Wohler's classic synthesis of urea (more than a hundred 
years ago) that some unknown vital principle sets apart 
the chemistry of living things from that of non-living. 

And yet there is a difference which expresses itself in the 
chemical make-up of the living thing. It is its organiza- 
tion. 1 The difference with respect to chemistry thus lies 
in the peculiar combination of compounds which together 
make a heterogeneous system. This acts as a unit-struc- 
ture, whose behavior or manifestations are those of a single 
thing and not the sum-total of the multitudinous chemical 
components in an agglomerate mass. 

Living matter has an organization peculiar to itself. 
Nowhere except in the living world does matter exhibit this 
organization. Life, even in the simplest animal or plant, 
so far as we know, never exists apart from it. Resting 
above and conditioned by non-living matter, life perhaps 
arose through the chance combination of the compounds 
which compose it. But who knows ? A living thing is not 
only structure but structure in motion. As static, it 
reveals the superlative combination of compounds of 
matter; as a moving event, it presents the most intricate 
time-pattern in nature. Life is exquisitely a time-thing, 
like music. And beyond the plane of life, out of infinite 
time may have come that harmony of motion which 
endowed the combination of compounds with life. 

Clearly then, the state of being alive reposing in com- 
binations, in the order in which the constituents are 

1 Dujardin, iSjj; Briicke, 1S61; Allmatu fS/g; Venvorn, iSgg; 
Moore, IQ2J. 



assembled both in space and in time, its investigation is 
limited. The direct analysis of the state of being alive 
must never go below the order of organization which charac- 
terizes life; it must confine itself to the combination of 
compounds in the life-unit, never descending to single com- 
pounds and, therefore, certainly never below these. 
Whereas physical science has to do finally with ultimate 
units of matter, the scope of biological research embraces 
the behavior of more heterogeneous combinations of these 
units. The physicist aims at the least, the indivisible, 
particle of matter. The study of the state of being alive is 
confined to that organization which is peculiar to it. It 
may be that life can never be written as a formula because 
it may be a physics and chemistry in a new dimension which, 
though superimposed upon the now known physics and 
chemistry, lies in an infinity which the human mind can 
not ever embrace — as a tone which theoretically we know 
exists but which the human ear can never hear. But be 
this as it may, life as an event lies in a combination of 
chemical stuffs exhibiting physical properties; and it is in 
this combination, i.e., its behavior and activities, and in it 
alone that we can seek life. A living thing represents in 
its unit of structure and behavior the highest order of com- 
plexity in nature. All this implies that the method 
employed in the investigation of a living thing can not be 
identical with that used in physical sciences. 

With right we glorify refined and precise measurements — 
great accomplishments in the physical sciences rest upon 
them. The desire to extend their use to embrace the science 
of living things is understandable; nevertheless, quantita- 
tive measurements can never be used in biology to the 
extent that they are used in physics. 

To be sure biology also employs measurements. He who 
studies the living animal or plant often has recourse, most 
of the time entirely unconsciously, to the estimation of 



weight and size. The classification of living things, the 
earliest scientific treatment of life, relies upon measure- 
ments. The anatomist, be he interested in bones or in 
chromosomes, reckons size and number. These kinds of 
simple and direct measuring have a definite place in biology. 
Similarly have those relating to the functions of the living 
thing. No one questions the value of measurement for the 
analysis of the gases in human blood. He who would be 
foolhardy enough to deny the dependence of biology upon 
physics and chemistry would do well to ponder the history 
of the research on animal respiration, beginning with 
Lavoisier, whose work stands as a new starting point for 
both chemistry and biology. The final proof that the blood 
carries most of its contained oxygen in chemical rather than 
in physical union constitutes one of the most brilliant chap- 
ters in biology to read which quickens the pulse. The 
measurements on temperature, on pressure, of electrical 
conditions, of light, of the chemical breaking down of com- 
plex foods and the building up of these, the photosynthetic 
process in green plants without which human life would 
become immediately impossible — these and many others 
show that biology comprises a welter of physico-chemical 
measurements, a fact to occasion no astonishment since 
living things are physico-chemical entities. 

The measurements employed in physical science which 
do not apply to living things concern states of matter below 
that level of organization which characterizes a living thing. 
True, matter in the living state as all matter is compounded 
of electrons. The high and enviable excellence of modern 
physics rests upon the beautiful and enthralling analysis 
of the ultimate unit-structure of the least particle of which 
all nature is composed. However, the fact that life as far 
as we know exists as a composite, and only as such, 
renders pure physical analysis, i.e., into electrons, inappli- 
cable to the state of being alive. The fact that life dissolves 



even when it is resolved only into compounds means, in the 
present state of our knowledge of electrons, an insuperable 
obstacle to an analysis of life into electrons. This would 
still be true were Ave suddenly to discover a peculiar com- 
pound which we could define as the compound of the state 
— or event — of being alive. As matter, a living thing may 
be analyzed with the means utilized in the resolution of 
non-living matter; but the moment we end the living state 
we render impossible any direct analysis of life. Clearly: if 
life exists only in a super-compound-state, contingent upon 
aggregation, analysis of its compounds being useless, 
analysis of molecular and atomic structure becomes equally 
futile. A quantitative biology that does not recognize this 
fact is doomed to failure. 

Without needlessly elaborating, I do nevertheless wish 
to make myself understood. It would be shamefully unfair 
and ungrateful of any biologist even to appear to discount 
the researches in chemistry upon which modern biology so 
largely rests. The greatest biological investigator of our 
time, Pasteur, was a chemist. His work indicates the 
extent to which chemistry may carry biology. The work 
of Emil Fischer, of Albrecht Kossel and of a host of other 
chemists has fructified and fortified biology. Still the fact 
remains: the exact analysis of the compounds which com- 
prise a living thing is only analysis of the compounds and 
by destroying life, such analysis fails to reveal the secret, 
the goal of all biology, the answer to the question, what is 

Here one may interpose a question, an inquiry concerning 

the postulated life-molecule. 1 In earlier times one thought 
of life as reposing in a peculiar molecule, as the biogen- 

molecule. Chemical researches have however so far failed 

to reveal the life-molecule. Instead they show that living 

1 See Hopkins, 1912. See also, Minot, 1S96. 



matter is composed of proteins, carbohydrates and lipins 
together with water, electrolytes and gases. It would be 
necessary, unless life resides only in protein, and this has 
not yet been proved, to obtain evidence to indicate that all 
these constituents named as components of living matter in 
some way combine as a single molecule. Further, with 
respect to proteins, carbohydrates and fats, we have no 
sufficient ground to warrant the conclusion that they exist 
in living matter in the same form which they have when 
they are isolated. This is especially true of the proteins; 
all of them known to us outside the living substance are 
probably changed in their nature chemically and physically 
by the methods of isolation. In the chemist's test-tube 
the organic constituents of the living substance may repre- 
sent merely end-products of something in the living. In 
this, however, lies no reason for supporting the theory of a 
biogen-molecule; rather should we try to refine our methods 
of Isolation, so that the results allow a more direct con- 
clusion. If one says that the biogen-molecule depends 
upon a certain milieu — if, for example, being protein-like 
it depends upon fat and fat-like substances, sugars, various 
electrolytes and water — we need the postulate that the life- 
molecule is totally unlike molecules with which physical 
science has to do. I have no wish to quarrel with Avords; 
either the term molecule used in the expression, life-mole- 
cule, carries the connotation ordinarily assigned it by 
physical scientists or it embodies a non-physical conception. 
As a concept consonant with physical science it stands open 
to serious doubt; as a non-physical postulate it defeats its 
own purpose. 

What is said of the biogen-molecule may be said of the 
gene-molecule, the hypothetical unit of which the chromo- 
somes are assumed to be built. Like the former it too 
appears incapable of self-maintenance — it grows by what 
it feeds upon, the cellular matrix in which it lies; and more, 
never stands apart from other genes in the same chromo- 



some. Since, according to the gene-theory, it represents 
in the body of the organism in which it exists a character 
which another gene (or genes) in another chromosome 
(or other chromosomes) may represent, it appears to act 
not singly but as part of the chromosome-structure and of 
the whole chromosome-garniture. We know that life 
exists in size below that recognizable by the microscope; but 
we possess no proof that life consists of a single gene. Much 
has been said recently of the size of the gene; but we should 
recall that size does not condition molecularity. Molecules 
vary in size. In addition, many investigators have become 
cautious in speaking of the gene as a molecule, preferring 
the idea that it may be several molecules. 

The most potent objection to the gene-molecule- (or 
molecules) concept as the unit of life lies in the fact that the 
gene-theory fails to explain how a single cell, the egg, 
becomes a complex animal. A detailed discussion of 
this point is given beyond. Here it suffices to say that 
a particle (or all the particles together) which is only 
in part engaged in the unfolding of life during its course 
from egg to adult can not constitute life itself. 1 The 
conception of a hypothetical life-molecule is barren and 
indicates again the limitation of the quantitative method 
of analyzing life below the level where life is. The investi- 
gator of the living state can and must use physics and 
chemistry since the living state is a zone in nature and 
his method of investigation can parallel that of the physical 
scientist inasmuch, as he finally comes to the unit-organi- 
zation of life. Below this he can not go, for life is the 
harmonious organization of events, the resultant of a 
communion of structures and reactions. 

In general, the organization of living matter, that is, of 
protoplasm, appears as consisting of two components, a 
nuclear and a cytoplasmic. Although most often these 

1 Bui cf. Jennings, 1936. 


are set off as two distinct regions, as a sphere (nucleus) 
within a sphere (cytoplasm), this sharp differentiation is 
not invariable. For several reasons, as will be shown 
beyond, much of modern biological investigation has 
centered upon the nuclear component as though it were 
indeed the kernel of life. Not only has the cytoplasmic 
component been relatively neglected but also have those 
protoplasmic systems which lack sharply defined and set- 
off nuclei received scant attention, although a bacterium 
whose protoplasmic organization fails to show a discrete 
nucleus is a living system. Because of the rapid rise of 
genetics, hegemony in the protoplasmic organization has 
been ascribed to the chromosomal structure of the nucleus 
and the cytoplasm has been subordinated as though it be a 
mere protective and nutritive shell. It is no part of the 
purpose of this book to minimize the achievements of 
genetics and the investigations on chromosome-structure, 
all outgrowths of descriptive studies on protoplasmic 
organization. Instead, inasmuch as life, as we know it so 
far, resides in the whole system, the pages which follow 
aim to show how far life-processes are related to the dual 
and reciprocal components, nuclear and cytoplasmic 

Investigations on cytoplasm have had largely to do with 
the formed bodies, the so-called cytoplasmic inclusions; 
only to a very small degree have they dealt with the cyto- 
plasm itself and its differentiation into an inner and an 
outer region, endoplasm and ectoplasm. Only in a general 
way has the role of the ectoplasm, the surface cytoplasm, 
been hinted at; thoroughgoing descriptions of it, compar- 
able to those of the nucleus, have not been made, doubtless 
because of the greater difficulties of observing it. None 
the less should the ectoplasm, as part of the protoplasmic 
system, be assigned a role in vital manifestations. An 
examination of its properties and behavior in life-processes 



is all the more imperative because it has been so grossly 

The play of environmental forces demands the continuous 
adjustment, balance and discrimination of the living thing. 
How this self-regulation comes about constitutes a great 
problem in biology. Not less capable is the power of self- 
differentiation exhibited by the living system. Against and 
with the outside world, repelling and responding, it makes 
itself anew, exhibits a series of events in sequence and 
order ever the same. Self-regulation and self-differentia- 
tion are fundamental expressions of the organization of 
living matter. 

As an integral part of the protoplasmic structure the 
ectoplasm can not be divorced from this. The proto- 
plasmic organization however much a composite of many 
parts — nucleus with its structures and cytoplasm with its 
regions — acts as a unit. To conceive it as such is to 
approach an understanding of those series of moving events 
that we call life. The ectoplasm, standing between the 
protoplasmic system's inner substance and the outside 
world, reacts first to environmental stimuli and thus condi- 
tions the responses of the whole system. Its rapidly occur- 
ring, highly active structural changes portray self-regula- 
tion and self-differentiation. Thus by its location and by 
its peculiar attributes, the ectoplasm becomes the most 
tangible expression of life-processes. 

Life a?id Rxperi??ie?it 

The method employed in the investigation of the 
living thing, the foregoing statements clearly indicate, 
must be different from that in physical sciences: the nature 
of the thing investigated determines the method of inquiry. 
Description is the method of most use in biology. It plays 
a larger role here than in physics not because experimental 
biology is a younger science than physics, as one often 
avers, but because of the heterogeneity and complexity of 
the composite life-unit. The more heterogeneous and 
complex an object, its parts undergoing multitudinous 
kaleidoscopic changes, the more important description 
becomes. And when this object is minute, its changes 
evanescent, accurate description is imperative. By 
description the objects may be defined and the changes 
recorded in more and more closely set stages. 

This statement, however, by no means implies that 
experiment has no place in the study of the state of being 
alive. Both description and experiment are utilized by 
all the natural sciences — the extent to which each is 
used being determined by the state of the matter investi- 
gated. Just as the complex organization of living matter 
demands the larger employment of description, so it 
prescribes experimental method. The present chapter 
aims to evaluate the place of experiment in the study 
of a living thing in order to clear the ground for the dis- 
cussion of the problems with which this book deals. Thus 
I present my conception of the role of experiment in 



biology, a conception which resulted from years of expe- 
rience and which by determining more and more my 
mode of experimenting had a considerable influence on the 
formation of my theory of the state of being alive. In order 
to set my conception off from that maintained by a sub- 
stantial number of biologists, I begin by defining their 
point of view. 

I refer to the so-called ''physico-chemical" school of 
biology. Specific example is often the best of definitions, 
I could give many examples from the writings of the 
" physico-chemical" biologists to define their position; one 
will suffice. Says Loeb 1 : 

The physical researches of the last ten years have put the 
atomistic theory of matter and electricity on a definite and 
in all probability permanent basis. We know the exact 
number of molecules in a given mass of any substance whose 
molecular weight is known to us, and we know the exact 
charge of a single electron. This permits us to state as the 
ultimate aim of the physical sciences the visualization of all 
phenomena in terms of groupings and displacements of ulti- 
mate particles, and since there is no discontinuity between 
the matter constituting the living and non-living world the 
goal of biology can be expressed in the same way. 

It is often said that in their thinking biologists are the 
most mechanistic of scientists, holding fast to concepts 
that the most mechanistic astronomers even have aban- 
doned. Indeed, many biologists besides Loeb believed — 
and many even now believe — that biology as a science must 
finally be concerned with the ultimate particles, that life 
as well as all physical phenomena can be interpreted on the 
mechanistic basis of the motion of particles in a system that 
is rigidly ordered as to time and space. But so far any 
attempt at "the visualization of all phenomena in terms of 
groupings and displacements of ultimate particles" has 

1 Loeb, 1916. 



failed for biology. And it is to be doubted that any such 
"visualization" can ever succeed. We do not know that 
there is no discontinuity between the non-living and the 
living world and we certainly possess no evidence for the 
postulate that living phenomena can be expressed in 
"terms of groupings and displacements of ultimate 

Nowadays, even for the physicist, Loeb's statement is 
too extreme. The evidence of physics does not yet permit 
such a view as to the finality of its concepts. In addition, 
before physical principles can be utilized profitably in 
biology, they must be sharply defined and accepted by 
physicists themselves. 1 Moreover, even before the advent 
of the relativity- and the quantum-theory, physicists 
were not agreed that mechanics constitutes all of physics. 
Here a statement written forty years ago by the biologist 
Whitman 2 is apt: 

While biology is certainly indebted to physics for some of 
its metaphysics, it is to the credit of physics to have made it 
clear that mechanism, indispensable as are its methods, 
affords no fundamental explanation of anything. As Karl 
Pearson has so well said, the mystery of life is "no less or no 
greater because a dance of organic corpuscles is at bottom a 
dance of inorganic atoms." What dances and why it 
dances is not explained by reducing size to the lowest limit of 
divisibility, and just as little by the assumption of ultra- 
physical causes. This is no criticism — no disparagement; 
it is only a confession of ignorance. The ultimate mystery 
is beyond reach of both mechanism and vitalism; let preten- 
sion be dropped, and approximation to truth be closer on 
both sides. 

Further, as we have seen in the preceding chapter, our 
investigations of the living thing as such end with its dis- 
integration. The moment that any such far-going physico- 

1 Watson, 1931. 

2 Whitman, 1S93. 



chemical analysis, as that postulated by Loeb, is applied, 
the living thing disappears and only a mere agglomerate of 
parts remains. The better this analysis proceeds and the 
greater its yield, the more completely does life vanish from 
the investigated living matter. The state of being alive 
is like a snowflake on a window-pane which disappears 
under the warm touch of an inquisitive child. 

This objection to the so-called physico-chemical point 
of view — i.e., that the goal of biology is the reduction of 
living matter to ultimate particles — is far more potent 
than those mentioned before. Certainly, it would still 
remain were the physicists able to state their concepts of the 
ultimate particles of matter in final terms. Though com- 
pounded of the same elements found elsewhere in nature and 
showing physical properties because of this composition, 
though amenable to physico-chemical laws, the living thing 
is refractory to analysis into ultimate particles. 

Here Heisenberg may be quoted to show how we can 
conceive the particular realm of living things as part of 
the natural world and the natural sciences: 

If for instance one thinks of the problems which are con- 
nected with the existence of living organisms, one will 
suppose, from the point of view of modern physics, that the 
powers which act in the organisms limit themselves in a like 
manner that is rationally exactly perceptible, from the 
purely physical laws, as, for example, thermodynamics 
limits itself from classic mechanics. . . . The building of 
natural science therefore most probably can not become a 
continuous unit, so that simply by following the prescribed 
way one can come from one point in it to all the other rooms 
of the building. Rather, the building is made up of single 
parts, each of which though standing in manifold relations 
to the others, nevertheless is a unit that is in itself complete. 
The step from already completed parts of the building to a 
newly discovered one or to one to be constructed demands 
always a mental action which can not be performed simply 
by developing farther that which already exists. 1 

1 Heisenberg^ 1934* 



The physico-chemical biologists, however, do not picture 
natural science as Heisenberg does. Rather, they visualize 
the problems of the living organism as occupying a room in 
a single apartment of the building of physics, that of 
mechanics. Their view of physics, far more restricted than 
that of physicists themselves, has therefore obfuscated both 
the methodology and the philosophy of biology. This is 
shown by their loose usage of the terms, mechanism, 
mechanical, mechanistic and even machine, as though these 
be interchangeable. 

In biology the term, mechanistic, is used as the antipode 
of vitalistic. Since practise has legitimatized this usage, 
there may be little reason to quarrel with it. Nevertheless 
T wish to point out that the physico-chemical school of 
biologists has erred in elevating the term, mechanistic, 
beyond the meaning assigned it by physicists. By their 
own doctrine the term should have the connotation assigned 
it by physics. The true antipode of mechanistic is non- 
mechanistic. The term, non-mechanistic, by no means 
implies vitalism. Not every physicist who opposes the 
mechanistic conception deems it necessary to support a 
non-physical, super-natural concept. Rather, he holds 
that the behavior of the ultimate particles of matter is not 
rigidly determined, perfectly predictable. Logically, those 
biologists who conceive vital processes as phenomena to be 
interpreted by physics, should adhere to concepts of physics. 
The physico-chemical biology should take cognizance of 
the fact that physics has grown beyond "classical physics. " 

Physico-chemical analysis into ultimate particles and 
the hypotheses derived from such work establish the fact of 
the existence of similarities between living and non-living. 
By virtue of its peculiar organization in space as well as in 
time, however, the living thing occupies a level in the 
natural world above that of chemical compounds. From 
this organization spring those characteristics by which we 



commonly distinguish a living thing from a non-living; both 
the organization and these characteristics should claim more 
attention from those — be they biologists, mathematicians, 
physicists or chemists — who study the living state, than 
they have up to now received. Having agreed that there 
exists no chemistry peculiar to living things and that 
physical properties are possessed by the living and by the 
non-living as well, we have remaining the task of evaluating 
the differences. 

It is not implied that only similarities have been studied 
and never the differences of the two regions. Nevertheless, 
those differences which set apart a living thing from a non- 
living should be studied more extensively as such. I can 
not see how they can be investigated by physico-chemical 
methods in the sense of Loeb, that is, by resolution into 
ultimate particles, by methods suitable for pure compounds 
used in the chemist's laboratory, or by any other that does 
not maintain the integrity of the living state. Biology 
should develop its theories by a method of work adapted to 
the peculiarities of the living thing and therefore quite 
distinct from those used in pure physics and pure chemistry. 
This statement does not imply that we should discard 
entirely for biology the use of physical and chemical means. 
Surely, no one would set himself against the use of the 
microscope or any other most refined apparatus, and of 
reagents, drugs, dyes, etc., the common equipment in the 
study of the living thing. Biologists count, measure and 
weigh and seek to detect cause and effect. But whatever 
means we employ should be adapted to that particular level 
which the living thing occupies in the natural world. 

In an investigation which aims to explain the state of 
being alive, the first prerequisite is the appreciation of the 
limits which circumscribe this state. In the utilization 
of physico-chemical means, then, we need to recognize the 
extent to which we change the living state; and, if we go 



beyond it, we must realize this fact. For this reason, the 
most definite knowledge of the manifestations of the normal 
living state becomes necessary. Experiments in biology, 
then, fall into three categories: 

i. Experiments on non-living systems. 

2. Experiments on killed living systems. 

3. Experiments on living systems. 

Number 1 includes experiments that furnish comparisons 
between living and non-living systems and that elucidate 
the result of cellular activity which expresses itself in secre- 
tion and excretion. Here belong physical models and 
artificial systems, as for example, semi-permeable mem- 
branes, suspensions of soap in water — used to imitate 
cleavage-patterns of the egg — colloidal solutions, passive 
iron wire — employed to illustrate nerve-conduction — etc. 
Also, here belong chemical analyses of substances that are 
produced by the living cell, as for example, hormones and 
digestive ferments and excretions, as urine, etc. 

Number 2 includes experiments on the chemical composi- 
tion of living matter, killed in the process of analysis. 
Though not experimental in the strict sense of the word, all 
histological and cytological studies on fixed tissues and cells 
are included in this category. When properly used, fixa- 
tion gives a faithful picture of the living and as such has 
great value for the study of it. 

Number 3 embraces experiments on the living system. 
These are of two kinds: those in which the normality 
remains unchanged and those in which it is altered. By 
means of the former, valuable data are collected by which 
indicia and criteria are established for supplementing and 
extending as well as interpreting observations and descrip- 
tions of vital processes such as respiration, etc. Such 
experiments are also of incalculable value for medicine. 
By the second type of experiments in this category we 
often succeed in making evident the roles of various factors 



by comparing their behavior in the altered living with that 
in the normal. 

This review makes it at once clear that experimenting 
in biology requires not only knowledge of physics and 
chemistry but also, and in no less degree, that of the normal 
living organism, the fundamental object of biological 
investigation. The following discussion centers around 
experiments on eggs. However, it applies also to work on 
other types of cells. 

The investigator must possess familiarity with the bio- 
logical system whose investigation he undertakes. The 
chemist demands pure standardized materials for his work 
and the physicist is at pains to be sure that his apparatus 
always performs perfectly; neither is satisfied with experi- 
ments contaminated by sources of error that can be 
eliminated. Just because of the reason that biological 
systems are not to be compared to " chemically pure" sub- 
stances and that their performance is not always one 
hundred per cent, perfect, the condition of the living thing 
studied should be as fully known as possible. However, a 
chemist who will use only purest chemicals will often in 
biological work use cells or organisms in poor or even mori- 
bund condition; the physicist who in the physical labora- 
tory demands perfection in apparatus, frequently is 
content with whatever cell or organism given him for 
biological research, no matter how it behaves. Unfor- 
tunately, it is all too true of many biologists, even of many 
old-fashioned ones, that they investigate living systems 
which are not in optimum condition. In the haste to make 
experiments, many find no time to learn the optimum 
condition of the system under study. Some do not 
care and are content to report results that vary from day 
to day, while others would not be able to distinguish a 
really normal egg or other living structure from an abnormal 
one. Indeed, it would seem by the manner in which many 



treat the organisms on which they work, " material '" 
as they call them, that they prefer their living systems in a 
debased and degraded condition. 

Surely, of all biologists the investigator who experiments 
on eggs needs to exercise most scrupulous care in order to be 
sure that the cells under investigation are in optimum 
condition. In the investigation of a system of organs, an 
organ or even a tissue, like nerve or muscle, although one 
can never be absolutely certain that all cells present are 
normal, the high number of cells that compose such a 
structure guarantees a high number of normally reacting 
cells and thus increases the probability of a normal response. 
In the case of eggs, one deals with structures which are 
single units, individuals that show each its own state of 
normality or abnormality. Furthermore, whilst it is true 
that out of each egg arise cell-complexes — tissues, organs, 
and organ-systems — these differ so much in degree of 
activity, in time of origin and in relation to each other in 
space, that the determination of the normality of the 
developing egg, made up at a given stage of a number of 
cells equivalent to that in a given tissue with which com- 
parison is made, is more difficult than in this tissue. The 
developing egg is normal only if all these different com- 
plexes present are normal. But this necessity of careful 
investigation of the grade of normality in eggs has one 
important compensation which makes the study of eggs so 
fruitful for biology. The behavior in laboratory experi- 
ments of tissues, e.g., nerve, muscle, or tissues in culture, 
removed from an organism may not at all be the same 
as that in the intact organism. That countless investiga- 
tors have obtained the same laboratory results speaks on 
the one side for the excellence of laboratory procedures, 
on the other for the wonderful capacity of tissues removed 
from an animal's body to withstand experimental treat- 



ment. Whilst the doubt as to the identity in behavior of 
excised and of intact tissues does not deny the value of 
studies on the former for laboratory exercises, it should be 
borne in mind that only rarely and with great difficulty can 
these tissues be experimented upon in situ (in their normal 
surroundings). There is doubt that results from experi- 
ments made on tissues in vitro (excised) could be obtained 
from such experiments made on tissues in situ. With the 
egg the situation is different. We can speak with more 
certainty concerning its normality for in many cases nature 
gives us the opportunity easily to study it cc in situ" This 
advantage of a study in situ is also offered by many proto- 
zoan cells. The control of normality in them is even less 
difficult than in eggs, because they lack, even those with 
most complex life-history, the potential diversity of the 
egg-cell realized as development unfolds; the protozoan 
cell reduplicates itself only. This difference, however, 
though showing the greater difficulty in the study of eggs, 
indicates the greater possibilities of such study. Valuable 
as studies on Protozoa doubtless are for both biology and 
medicine, they lack direct bearing on the grand problem 
of biology, how out of a single egg arises the complex multi- 
cellular organism. Only in the egg and its development 
can we hope to trace to its source the pattern of structure, 
and to resolve into its motif the harmonious behavior 
which characterizes the many-celled animals. 

If the condition of the eggs is not taken into account, the 
results obtained by the use of sub-normal eggs in experi- 
ments may be due wholly or in part to the poor physiologi- 
cal condition of the eggs. Thus, the failure of sea-urchin's 
eggs that are freed of their jelly, fully to separate their 
vitelline membrane after fertilization, as they normally do, 
does not mean that the experimental removal of jelly 
renders membrane-separation impossible but only that the 


y/*°* *<*: 


eggs are in a bad condition brought about by the injurious 
action of the agent employed to remove the jelly. 1 The 
physiological condition of all eggs known to me can be 
impaired by exposure to low temperatures. Indeed, since 
low temperature (like high) is an experimental means, to 
experiment on eggs from animals which have been kept in 
the ice-chest in order to delay shedding, is equivalent to 
compounding experimental procedures whose effects may 
be complementary or antagonistic. Thus, I would never 
think of exposing eggs of Platynereis, for example, which 
had been kept over night in the cold room (at 5°C.) to 
ultraviolet light because the low temperature alone gives 
the effect of polyploidy to obtain which I use the ultra- 
violet radiation. 2 

From a series of experiments made on the capacity of a 
sea-urchin's eggs to develop without spermatozoa, it was 
concluded that development can be caused by immersing 
the eggs in sea-water which had been charged with a sub- 
stance liberated by others of the same species. This con- 
clusion was unwarranted because, as I found, the results 
obtained are solely due to evaporation of the sea-water 
and not to the presence of substances originating from the 
eggs. 3 Another series of experiments was used to prove 
that this subtance liberated by the eggs and held to be the 
cause of their development, had been isolated. But I 
found that sea-water treated by one or by all of the reagents 
employed for precipitating the alleged substance was if 
anything more effective for causing development than 
sea-water containing the egg-substance. Thus the develop- 
ment was due not to the isolated (precipitated) sub- 

1 For discussion of this subject, see Just, 1928c. Derbes, 1S47 
knew not only that jelly surrounds the sea-urchin's egg but also that 
its absence does not impair the egg's development. 

2 Just, /Q2Qe. 

3 Just, 1928a. 



stance liberated by the eggs, but to increased salinity of 
the sea-water. 1 

Few authors who quote Wilson's famous experiments on 
the eggs of the marine mollusc, Patella^ I think, realize that 
Wilson never succeeded in obtaining normal fertilization. 2 
It is not improbable that the results he obtained, upon 
which he based far-reaching conclusions which have been 
generally accepted, were in part due to the abnormality 
invoked by the artificial aid with which he induced fertiliza- 
tion. To induce cross-fertilization (fertilization of eggs 
with non-specific spermatozoa) often one has recourse to 
artificial aid, as changes in alkalinity or temperature of the 
sea-water. Such changes however bring about variations 
in the development of the egg (for instance of the sea- 
urchin) also if these develop from straight fertilization 
(fertilization of eggs with specific spermatozoa) . This 
fact makes it clear that results obtained after cross-fertiliza- 
tion can not be ascribed to the influence of the non-specific 
spermatozoon or its chromosomes unless it is shown that 
the effect differs from that obtained in straight fertilized 
eggs that were subjected to the same changes in the medium 
by which the cross-fertilization was induced. Since the 
experiments on cross-fertilization that have been made were 
not controlled in this way, they allow no conclusion as 
to the action of the foreign sperm-nucleus. 3 

Even under constant external conditions the sea-urchin's 
larval skeleton will vary, as most exact study of normal 
straight fertilized eggs has shown. 4 Thus the extent to 

1 Just, igzgd. 

2 Wilson, 1904. 

3 It must therefore astonish us that Morgan {1932) not only 
accepts these experiments. on cross-fertilization but also in a zvide- 
sioeeping statement categorically declares them to be proof for action 
of the genes on the cytoplasm. 

4 Tennent, 1910. 



which eggs in optimum physiological condition vary must 
also be known in order that differences discovered can be 
truly attributed to the experimental means. 

These few examples show how careful we must be in 
drawing conclusions from experiments in biology. The 
highly irritable systems that we investigate demand a most 
exact control of the experimental factors. If only one of 
these is left aside in our evaluation of the obtained results, 
our conclusion may be wholly erroneous, especially if the 
factor induced a change at the beginning of the treatment. 
We must know whether we have before us normal or already 
changed, abnormal, eggs. We must control the effect of 
our means, even of those that are only aids, by following 
their effect singly before we use them together in one experi- 
mental setting. And above all must we be wholly familiar 
with the normal condition and normal development of the 
eggs we use, since only on this basis are we able to recognize 
abnormalities and thus the effect or effects of our experi- 
mental treatments. 

The simplest and most generally adopted criterion for 
the eggs 5 normality lies in estimating the number of them 
that develop; one hundred per cent, development means 
optimum condition. Eggs that are laid already fertilized, 
and especially those in brood pouches or in capsules almost 
always develop normally. Also where the eggs and 
spermatozoa are deposited and fertilization ensues in the 
laboratory as in nature, the chances for normal develop- 
ment are enhanced. It is with those eggs whose deposition 
is induced by the experimenter and which are artificially 
inseminated and especially with those that are removed 
from the ovaries that most difficulties are encountered. 
In many cases such eggs develop, if at all, only in small 
numbers. They are only useful for studies on development 
if the cause for the low numbers of development can be 
discovered and the percentage increased. There are cases, 



however, where eggs removed from the animal yield one 
hundred per cent, development. Here it is necessary to 
know that the quality of development parallels that of 
eggs normally laid under natural conditions. By compar- 
ing the development of eggs in their normal environment, 
of those naturally shed in the laboratory and of those 
removed from the animal, one can decide concerning the 
normality of the last named. 

In rare cases, where always some immature eggs incap- 
able of development are present, one may never obtain 
large numbers of developing eggs, yet those that develop 
are normal. Similarly, at the end of a breeding season 
animals frequently give, among those eggs that develop 
perfectly, some that fail to develop because they have 
passed the time of optimum condition for fertilization. 
For such eggs we can not use the criterion of the percentage 
of development to detect their condition; they can only 
then be considered to be wholly normal, if their develop- 
ment is normal at every stage. 

Now the necessity of following the egg throughout its 
complete development can be obviated. I was able to 
establish definite criteria and simple physiological indicia of 
the optimum condition of eggs. These signs tell us within 
the first minutes after fertilization whether the eggs are in 
optimum condition and even indicate whether the eggs 
will be normal throughout their development. The signs 
inhere in the reactions following the mixing of eggs and 
spermatozoa and are in a measure as specific as the given 
gametes themselves. For any given egg, they appear 
differently when the egg is abnormal. For the eggs of a 
common sea-urchin, Arbacia, I found that their optimum 
condition, whether they are normally shed, induced to be 
shed by artificial means, or removed from the ovaries, can 
be determined within three minutes after insemination by 
the rate and quality of membrane-separation; by the 



rapidity with which unfertilized eggs separate fully their 
membranes while in distilled water; by the rate at which 
fertilized eggs form extra-ovates when in hypotonic sea- 
water; and by the failure of heavily inseminated eggs 
to show polyspermy. Whenever these initial reactions are 
qualitatively poor, the development of the eggs is below 
normal. 1 These criteria I find hold for three other species 
of sea-urchins at Naples. 2 Also, for the egg of Nereis the 
quality of the striking ectoplasmic changes subsequent to 
fertilization constitutes an index of the condition of the 
egg and the quality of its later development. 3 

Further I found criteria which in later stages of develop- 
ment indicate whether this will be normal in its whole 
course. For example, in eggs of Nereis and Platynereis, 
the behavior of the oil drops is a reliable index of normality; 
one may be sure that swimming forms which possess one 
oil-drop and only one in each of the four gut cells can 
develop through metamorphosis to the adult stage and 
that no other will. 4 Doubtless other criteria can be found 
for every stage of the development. Every one such will 
prove valuable in extending our knowledge as to what is a 
good egg. In all cases however, where such criteria are not 
known, the whole course of development must be always 
followed before it can be said that development is normal. 

It is clear that the condition of eggs largely depends - 
upon that of the animals from which they come. Eggs 
normally shed from animals in a good state are preferable 
to those shed by weak animals and by animals in abnor- 
mal conditions. In every experiment comparison with 
and control by the normal, untreated cell or organism 
is obviously absolutely necessary. The quality of the 

1 Just, 1928c. 

2 Just, 1929c. 

3 Just, I9i5 a - 

4 Just, I922g. 



investigated system determines the experimental results 
to such a high degree that the value of the investigation 
depends upon recognizing and appreciating this quality of 
the object. 

The most exact knowledge of the normal form and form- 
changes of the living thing to be investigated is thus the 
prerequisite for present-day attack of biological problems. 
On the strengthening of our knowledge of these rests all 
progress of modern biological research, no matter how 
grandly physical, chemical, or mathematical it is. 

For a long time to come biology will need accurate 
description and exact observation. The necessity for 
confirming the classic and exact studies still remains; it is 
imperative that these be extended. The demand for filling 
in gaps persists. Where minute details are wanting, they 
must be supplied. Wherever uncertainty or doubt 
obtrudes concerning a descriptive datum, this should as 
far as possible be removed. However much we desire 
quantitative instead of qualitative studies in biology, 
however much more we estimate elaborate experimental 
studies involving knowledge and skill in the use of physics 
and chemistry and mathematics, however much we yearn 
to place biology in the same category with the more exact 
sciences, we can not abandon purely descriptive work. I 
-do not mean, I repeat, that biology stands irreconcilably 
apart from the other natural sciences. But I see no omen 
to indicate that all biological phenomena are capable of 
quantitative treatment. 1 By chance to-morrow or it 
may be in the very instant of this writing by some great 
discovery made in total ignorance of the morphological 
substratum of biology, someone might be able to appreciate 
the secret of life in its entirety. But it is just as likely that 
this biological millenium may never come. And I for my 

1 Cf. Mellor, 1922, on the use of mathematics in science. 

2 5 


part believe that fully to embrace this faith in a chance 
discovery would anaesthetize activity and the will to seek, 
while life lasts, the mystery of life. 

That we are far from having completely exhausted the 
possibilities of description in biology is clearly shown by 
the fact that no one of the grand phenomena treated in this 
book has been adequately defined by description that fulfils 
our demands for exactness* 

My own observations having early taught me how rapidly 
changes ensue in the living egg, I have studied stages in 
development not of minutes' but of seconds' duration. 
Microscopy by extending the range of our vision increases 
our powers of observation. But even if there should be 
revealed to us the ultimate space-pattern, there would 
still remain the problem of the changes of this pattern in 
time; a gap between the beginning and the end of an event 
would persist. Just as with the aid of the various kinds 
of microscopes 1 we uncover the minutest space, so must 
we register most minute changes in time: we must clock 
the fleeting changes whose sum total is the living thing. 

Studies of forms and form-changes must continue as long 
as there are organisms and their processes, eggs and their 
development, still unexplored. Since not all work ever 
done in this field is wholly dependable and perfect, there is 
all the more reason why the modern student experimenting 
on eggs must acquaint himself directly with the pattern 
which he aims to explain. Naturally, in that earlier period 
in the history of the study of eggs the new findings, as 
fertilization, chromosome behavior, etc., excited and stimu- 
lated the discoverers to exuberant expressions that were often 
in error because they came from work which though 
honestly well done was not always sufficient to justify the 
conclusions drawn from it. Work like that of Boveri which 

1 Note the recently invented electron-super microscope. 



has given a great impetus to biology, but which nevertheless 
does not satisfy our demand for exactness and completeness, 
should most certainly be repeated on a more extensive 

Even to-day exactness is often not carried as far as we 
should demand. Above I gave examples of incompletely 
controlled experimental work. Where living cells are 
treated statistically, the larger the number used, the more 
valuable are the conclusions drawn. Few workers would 
care to publish results like some of Morgan's who in eleven 
experiments had i, 10, 4, 8, 8, 3, 3, 1, 2, 2, 4 eggs, and in 
other experiments had no eggs, yet spoke of percentage of 
development. 1 

Certainly, modern technique offers its advantageous 
apparatus also to biology for increasing exactness in 
observation and experiment. These modern achievements 
on the other hand have served in devaluating morphological 
knowledge, and impressed by technical progress many an 
investigator regards experimentation as a virtue in itself, 
a reward of its own. 

Experiment for experiment's sake, ever a dangerous 
philosophy, becomes exceedingly baneful for biology. 
True, through it valuable knowledge may be gained for 
mapping out unknown terrain. On the other hand, by it 
the accumulation of data may become bewildering because 
it relates to topographical details impossible of reduction 
on a scale of value for other explorers. Moreover, such 
refined plotting of points often has no relation to the whole 
field — indeed, often the field as such is lost sight of and only 
unrelated minutiae remain. The main purpose of an 
experiment in biology should be the explanation of the 
naturally occurring phenomena. Here we encounter a 
difficulty not met with in physical science. 

1 Morgan, 1905. 



Animal cells are ready-made. We do not devise them; 
nor can we have them according to specifications which we 
ourselves set up. If, as some do, we regard living cells as 
machines, we appreciate that they are ready-made; we do 
not make them as physicists make their apparatus to prove 
a theory or test an hypothesis. Still less do we know them 
and their variables. We do not use them to prove theories; 
rather, either we elaborate theories from our observations 
on them, or, having set up a theory, attempt to establish 
it by experiment. In either case we seek to know the 
" machine" and not by machine-making to devise hypothe- 
sis or establish theory. 

A cell is never a tool. Nor is it an instrument on which 
to whet one's physics and chemistry. Living matter is 
never an excuse and living phenomenon never an oppor- 
tunity for the display of the investigator's physico-chemical 
knowledge. If we use an apparatus in order to determine 
oxidation in an inanimate system or devise a sensitive 
instrument to measure light, the apparatus or the instru- 
ment is a tool; but if one determines oxidation in a cell, 
the oxidation-determination is the tool; if one measures 
light produced by a cell, the measurement is the tool. In 
neither case is the cell a tool unless one frankly wants to 
compare its sensitivity for oxidation or light production 
with that of a man-made machine which of course is an 
entirely different reason from that of investigating the 
properties named as peculiarities of a living thing. The 
physicist devises a tool with which to measure a given 
change or to test an hypothesis; the biologist tries to mea- 
sure changes in the cell — and the measurements are then 
the tool by which he evaluates the properties of the cell or 
tests his postulates of what these properties are. 

I think that we can agree that in the experimental study 
of the egg's development the aim is the analysis of the 
stages found in nature through which the egg passes to 



attain the normal adult condition. By experiment we 
attempt to ascertain the meaning of normally occurring 
stages which are co-existing and successive, co-ordinate and 
subordinate. We seek to unravel the tangle of the pro- 
cesses or, from another point of view, to stem the rush of 
developmental events in order to determine what in them 
is cause and what effect. The picture that the normal 
developmental process gives us must we trace in its every 
line and shadow and by experiment aim to elucidate their 

Hence, the method for the study of living systems is this: 
to extend to the utmost a purely biological attack — that is, 
to know qualitatively every process; to mark its beginning 
and its end and in its duration to map out most closely set 
phases; to chart its course. This appreciation of the 
normal life-processes will set the goal which we strive to 
achieve by means of experimental attack. No matter how 
far the experimental methods change normal condition and 
behavior, always should results obtained with them have 
bearing on the normal. The greater the extent of the 
change, the more surely must we realize that it is a change. 
Conclusions drawn from greatly altered states concerning 
the normal need most careful scrutiny. Death-changes 
induced by experiment should be appreciated as such and 
be considered as revealing the living condition, which 
they extinguish, in no other way except by extinguishing it. 

Those experiments which alter a normal process least 
have to-day especially great value in the study of the egg 
and its development. Few investigators nowadays, I 
think, subscribe to the naive but seriously meant compari- 
son once made by an eminent authority in biology, namely, 
that the experimenter on an egg seeks to know its develop- 
ment by wrecking it, as one wrecks a train for understand- 
ing its mechanism; he might also have said, as a young child 
breaks up a watch to see the wheels go around. The days 



of experimental embryology as a punitive expedition against 
the egg, let us hope, have passed. Instead comes the time 
of nice, exact, carefully graded treatment, wherever possible 
reversible in its action, for the purpose of analyzing develop- 
ment in ever more closely set stages, after this has been 
mapped out in the perfectly normal egg with meticulous 
care and scrupulous cleanliness. The most minute space- 
time organization of the living system makes the instrument 
on whose strings play the processes by action and inter- 
action. By experiment we here slightly exaggerate, there 
lightly fret the tones out of which the harmony of the 
living state arises. 



The Protoplasmic System 


not always reveal itself as consisting of one nucleus and 
surrounding cytoplasmic mass, though this type is very 
widespread in occurrence. There are living things in which 
a discrete nucleus can not be discerned and on the other side 
we know formations which show many nuclei embedded in 
one continuous cytoplasmic mass. Thus, we may distin- 
guish three types of the protoplasmic system according to 
the extent to which the nuclear substance is differentiated. 
A classification of the types of the protoplasmic system 
could also be made on the basis of the degree of the differ- 
entiation of the ectoplasm. But inasmuch as nuclear 
differentiation has up to now been more thoroughly studied, 
it is chosen here as the basis for a classification. 

Some bacteria and blue-green algae have been reported as 
non-nuclear organisms. In them nuclear substance may, 
however, be deposited as granules so finely subdivided that 
they escape detection. 1 Indeed, in some cases whilst no 
formed nuclei or nuclear areas could be located, the presence 

1 The presence of nucleic acid in bacteria has been reported. 
Schaffer, Folkoff and S. Bayne- Jones {1922) extracted from JOO 
grams of dehydrated bacteria grown on a synthetic medium free from 
purine and pyrimidine compounds {constituents of nucleic acid) a 
non-hygroscopic, protein-free, powder which they consider a nucleic 
acid, though it contained no pentose, the sugar of plant nucleic acid. 
In the opinion of JF. Jones and Folkoff {1922) the substance is a 
nucleic acid. 



of granules has been demonstrated, which respond to 
reagents as does chromatin (a substance found in nuclei). 
In others, by means of tests with dyes, chemical reagents, 
etc., investigators have demonstrated areas which respond 
as do nuclei in larger cells. Such systems are said to 
possess diffuse nuclei. 

So much is clear: there are living forms which show no 
discrete nuclei. This fact indicates that in the organiza- 
tion of protoplasm the presence of a discrete nucleus is not 

The blue-green algae — bacteria are very closely related 
to them — evoke special interest because they probably 
represent the first forms of plant-life that arose on the earth. 
If it be established that there exist non-nucleated blue-green 
algae, this might mean that in the emergence of life, nucleus 
and cytoplasm were undifferentiated and only subsequently 
in the course of evolution became separated. 

Doubtless there exists living matter of such minute size 
that it can not be seen with the highest powers of the 
microscope. Whether or not such protoplasmic systems 
possess the organization exhibited by cells seen under the 
microscope remains to be learned. In a discussion of the 
visible form of organisms it would be futile to include 
invisible forms. Nor can ultra-filterable viruses be 
included. In addition to the fact that they are of submicro- 
scopic size is the question whether they be living things or 
only the product of living things. If they be living, they 
may despite their minute size possess the nucleo-cytoplasmic 
organization of visible cells; or, lacking such they may 
prove to be the most elementary protoplasmic system — 
protoplasmic ground-substance without clearly differen- 
tiated regions. 

Many organisms consist of a mass of cytoplasm contain- 
ing two or more nuclei. These may be low forms of life, 
Protozoa, like the slime-mould or the multi-nucleated 



Opalina. Also among the higher animals certain sheets of 
protoplasm as the binding or sustaining (connective) 
tissue or the heart muscles of vertebrates show innumerable 
nuclei throughout their extent without interposed cell- 
boundaries. Such multi-nuclear protoplasmic systems are 
called syncytia. 

The existence of syncytial protoplasmic systems has 
given rise to theories opposed to the so-called cell-theory 
which postulates that the cell is the unit of structure and of 
function. Rohde's views 1 concerning the preeminence of 
syncytia may be dismissed because they have little founda- 
tion in fact. Similarly, Studnicka's arguments 2 as to the 
living nature of cellular formations, fibres, etc., which 
come to be extra-cellular, do not warrant discussion here. 
The argument set up by Whitman 3 and by Sedgwick/ 
that the cell-theory of development is inadequate, out of 
which have grown the so-called organismal and organism- 
as-a-whole conceptions merits more attention although it 
too has slight basis in fact and is indeed almost wholly 

These conceptions originated as protests against the 
extremist's point of view that the individual cell is the 
end-all and be-all of life even in complex organisms; that 
through exact knowledge of single cells one could win an 
explanation of the organism as a unit. To-day we appre- 
ciate the fact that a mere agglomerate of cells equal in 
number to those that constitute an organism is not an 
organism. That organisms are units and act as such, be 
they composed of single cells, as Protozoa, or of myriads 
of cells, as the higher animals, no one will deny. The 
organismal or organism-as-a-whole conceptions have ren- 

1 Rohde, 1923. 

2 Studnicka, 1934* 

3 Whitman ) 1S93. 

4 Sedgzvick, 1892. 



dered some service therefore in emphasizing this fact so 
universally accepted. Enough is known concerning the 
difference in behavior between cells when en rapport with 
others that make up the intact organism and those having 
been isolated thereform, concerning the development of 
eggs and the significance of the orderly sequence in time 
and in space when specific embryonic cells are set off and 
concerning the pathological growths, as tumors, to warrant 
the clear conclusion that cells alone and as members of a 
unit-system exhibit different behaviors. However, no suffi- 
cient reason exists for assigning to cellular differentiation, 
the integration of cell with cell, or the specificity of organ- 
isms which resides only in cells and their products — all well 
accepted biological truisms — the new terms of organismal 
or organism-as-a-whole. 1 Moreover, the principle of unity 
— the expression of integration, differentiation and of 
specificity both in form and behavior- — which distinguishes 
an organism from a mere mass of like cells, ought not be 
elevated to the position of an abstract principle divorced 
from the concrete physical basis on which living things are 
organized. 2 

An organism possessing no formed nucleus represents 
one, and a mass of cytoplasm containing many nuclei 
represents the other extreme type of protoplasmic systems. 
If we assume that nuclear structure (and therefore from 
it the discrete nucleus) is a derivative of the ground- 
substance, the various types of protoplasmic systems can 
be reconciled: the single nucleus of a simple cell is then 
the compounding of nuclear bodies evident in non-nuclear 

1 In view of the claims of priority pressed by several writers 
it may be worth-while to note that Descartes {1662) conceived 
the organism as a whole. See Delage's {1S95) discussion of 
" organicisme" 

2 See Woodger^ 1929, / 9J° an ^ / 93 / if or recent discussion of the 
organism-as-a-whole point of view. 



forms like bacteria; the compounding of nuclei themselves 
(a result easily obtained experimentally in normally mono- 
nuclear cells) leads to the multi-nucleate, syncytial or 
polyenergid condition. To a consideration of that proto- 
plasmic system which is most widespread in occurrence and 
which reveals itself as a simple membrane-enclosed cell 
with a single nucleus, I now turn. 

For most organisms in both the animal and the plant 
kingdom this cell is the basic structural unit, no matter 
how complex or how large such an animal or plant may be. 
A whale and a giant red-wood tree as well as minute forms 
like a vinegar eel and a thread of green alga are patterns 
of cells. Animals and plants also exist as single cells, 
which within their boundary complete their life-cycles, 
exhibiting the vital activities shown by the more complexly 
organized individuals. These activities are perfect; diges- 
tion, respiration, conduction, contraction, reproduction, in 
a unicellular organism are as complete, though not so 
complex, as in man. These individuals in their organiza- 
tion and behavior reveal the single cell as an independent 
unit. At first one is amazed that microscopic creatures like 
the slipper-animalcule and the diatom, a plant of micro- 
scopic size, can carry on the functions of more highly 
organized forms of life; they seem at first to be imitations 
of the latter. But observation teaches that the millions of 
cells which make up the body of a multicellular animal or 
plant have lost in capacity: no one cell in them can perform 
all the functions to the same degree that a single-celled 
individual can carry out. 

This loss of capacity is compensated for by an emphasis 
on some one function, as shown, for example, in cells in 
the human body. In it some parts, which together make up 
the nervous system, take over the business of conduction 
which is developed to a high degree. Other parts, the 
muscles, emphasize contraction ; still others, respiration, 



digestion, excretion, reproduction. Thus we speak of the 
various systems — nervous, muscular, respiratory, etc. 
Each system is made up of organs; each organ is a combi- 
nation of different tissues; each tissue is a collection of cells 
of the same origin and usually of the same form. 

Out of a single cell, the egg, emerges all this complexity 
of organization found in the adult human being or in most 
other multicellular organisms, animal or plant. The human 
egg during its development forms tissues, these aggregate 
to form organs, and the organs are united into systems. 
In an animal's origin, as well as in its definite adult struc- 
ture, therefore, the cell is the unit of organization. This 
fact, together with that of the existence of unicellular 
animals and plants, shows that a large part of the world 
of living things is organized on the basis of cell-structure. 

The history of the multicellular organism as it develops 
from the egg, a single cell, to the adult is very much like a 
synopsis of the history of the whole world of multicellular 
organisms; this has most probably evolved from a single- 
cell ancestor. Thus biologists classify animals and plants 
beginning with the single-cell individuals and ascend- 
ing through grades of increasing complexity. Among 
animals, for example, the Protozoa stand first; next are the 
sponges, animals possessed of tissues only; above the 
sponges stand animals whose tissues are organized into 
organs and finally come animals with systems of organs. 
From the point of view of the history, both of the individual 
multicellular organism and of the world of multicellular 
organisms, the cell is the unit of the state of being alive. 

That this cell must be regarded as a unit becomes further 
evident if we consider the fact that nucleus without cyto- 
plasm or cytoplasm without nucleus is incapable of living. 
It is true that pieces of cytoplasm devoid of nuclei, as the 
red blood corpuscles of mammals, occur normally and live 
for a time; but these are specialized structures incapable of 



the full complement of living functions. One can cut off 
pieces of a single-cell animal; if these pieces be without 
nuclei they die, whereas a piece containing the nucleus or 
pieces containing portions of the nucleus are capable of 
growth to the full size of the normal creature. In turn, 
nuclei deprived of their cytoplasm do not live. The unit of 
living matter in these cases is thus shown to reside in the 
nucleo-cytoplasmic organization. 

We therefore adopt the point of view that the proto- 
plasmic system is the unit of life. For the majority of 
animals and for all their eggs the protoplasmic system 
reveals itself as comprised of a single nucleus enclosed by 
cytoplasm — ground-substance containing suspended mat- 
ter, the cytoplasmic inclusions — -and the whole enclosed 
by a membrane. Such cells exhibit variations in several 

Cells may show extreme size-variations. They range 
in size from one micron in length, discernible only under the 
highest microscopical magnification, to the sporozoon 
parasite found in the digestive tract of Crustacea which 
measures 16 mm. in length, or to the unfertilized ^gg of the 
ostrich, 105 mm. in diameter, or that of a shark, 220 mm. 
in diameter, or to the nerve-cells in the human spinal cord, 
which may be one meter in length. Organization of the 
cell thus does not depend upon size. We may imagine that 
the skein of life is greatly contracted in some while it is 
more diffuse in others. The maintenance of the cell-size 
that is characteristic of a species still remains an unsettled 
problem. Maintenance of a definite and specific size is a 
revelation of the self-regulative capacity k of the proto- 
plasmic system. 

Cells also show most varied shapes and forms; some are 
almost perfect geometrical figures — spheres, cylinders, 
cones; others are irregular or of forms not easily reducible 
to conventional geometrical figures. Often when in groups 



they show the effect of compression; thus cells which, were 
they free, would be spheres, become polyhedral. The 
varied shapes of cells depend upon many diverse factors in 
addition to compression, such as presence of a supporting 
frame-work, as in Foraminifera, the greater or lesser fluidity 
of the cytoplasm, the structure of the limiting surface, and 
most of all the little understood intrinsic structure of the 
cell itself. 

Cells, like all matter, are chemical in composition and 
display physical properties. Their form, structure, and 
morphology, however varied, are molecular in make-up. 
Hence, as we have seen, there is, in this respect, no dif- 
ference between living and non-living matter. All the 
protoplasms known to us have in common a certain 
basic chemical structure. Since living things are part of 
the natural world it is not astonishing that they are com- 
posed chiefly of those chemical elements which are most 
frequently found in the world of non-living things: Carbon 
(C), nitrogen (N), oxygen (0), hydrogen (H), sulphur (S), 
phosphorus (P), iron (Fe), sodium (Na), potassium (K), 
calcium (Ca), magnesium (Mg) and chlorine (CI). In 
some cells are found other elements as lithium, barium and 
strontium; copper and zinc; fluorine, arsenic, bromine and 
iodine. Thus the difference between non-living and living 
systems does not reside in the basic elements of which they 
are composed. A difference obtains in the compounds into 
which these elements enter. 

The elements, C, H, N, O, combined build up the protein 
molecules; out of C, O, H carbohydrates are formed; and 
C, O, H makes up most lipins; other lipins in addition 
contain N or P. These are the "organic" compounds of 
the living substance and exist in nature only in living sub- 
stance; non-living things do not contain proteins or the 
oils found in animals and plants; sugars and starches 
(carbohvdrates) occur naturally only in animal and plant 



tissues. Thus, not the elements as such characterize the 
structure of living matter but the peculiar combination of 
elements which constitute the protein, the lipin and the 
carbohydrate molecules of living matter. 

These compounds are suspended and dissolved in an 
aqueous medium containing electrolytes as Na, K, Ca,Mg, 
CI, etc., with oxygen and carbon-dioxide. The whole mass 
of protoplasm — organic compounds, electrolytes, water and 
gases — it seems, maintains a fairly constant slightly alkaline 

Each of the three organic compounds is built up of 
simpler molecules strung together: the proteins are strings 
of amino acids formed in some such manner as the following: 

C 4 H 9 .CH(NH 2 ).CO.NH.CH 2 .CO.NH.CH 2 .COOH 

which is a poly-amino-acid or tripeptide of leucin: 

CH 3 CH.CH 2 .CH(NH 2 )COOH 

and two molecules of glycin: 

CH 3 CH 2 .NH 2 .COOH 

The more complex proteins show comparable linkage. 1 

The lipins, as shown by the chemical structure of a simple 
fat, are combinations of three molecules of fatty acid and 
one of glycerol, as: 

CH3.CH2.CH2.COOH + CH 2 .OH 
CH 3 .CH 2 .CH 2 .COOH + CHOH 
CH 3 .CH 2 .CH 2 .COOH + CH 2 .OH 

with the loss of three molecules of water. 

Monosaccharides (simple sugars), disaccharides (second- 
ary sugars) and polysaccharides (starches and the like) 

1 With the aid of the ultra-centrifuge and by means of x-ray- 
spectroscopy valuable information is to-day being gathered con- 
cerning the protein-molecule. 



comprise the carbohydrates. Simple sugars like dextrose 
have the formula, C 6 .Hi 2 .06, secondary sugars 2(C 6 H ]2 06) 
— H 2 0, or Ci 2 H 22 0n, whilst more complex carbohydrates 
are only more molecules of simple sugars piled up. 

Whereas plants because of their photosynthetic power 
can in sunlight or in artificial light produce sugars from C0 2 
and H 2 in the presence of the green substance, chlorophyl, 
and from sugar thus formed synthesize lipins and proteins 
when X is present, most animals ingest food as protein, 
carbohydrate and lipin. Before utilization by the animal 
organism, these compounds must be broken down, i.e., 
digested, into their component parts: proteins to amino 
acids, carbohydrates to dextrose and laevulose and lipins 
to fatty acids and glycerol. These fractions are built up 
again by the organism into the more complex molecules. 
Special significance is attached to the synthesis of amino 
acids, for the proteins thus formed by the organism are 
peculiar to it and not to the organism which furnished them; 
thus herbivorous animals, cattle for example, convert the 
plant proteins of their food into proteins peculiar to beef. 

The break-down of these compounds is accomplished by 
enzymes, chemical bodies which act much as catalysts- 
i.e., by accelerating chemical changes. Enzymes show a 
high degree of specificity. For proteins, there exist the 
enzymes like pepsin found in the human stomach and 
tripsinogen, produced by the pancreas; for the starches 
is the enzyme, amylase; for the sugars, sucrose, maltose, 
lactose, the enzymes are sucrase, maltase, and lactase 
respectively; for the lipins, lipase. The break-down is 
one in which water is used, a so-called hydrolytic cleavage. 
Enzymes also synthesize the end-products of digestion into 
proteins, carbohydrates and lipins in which case water is 
lost. Some enzyme-reactions are reversible: thus, the 
reaction lipin plus lipase = fatty acid plus glycerol, or 



fatty acid plus lipase = lipin — depending upon the amount 
of water present. 1 

The chemical make-up of the three organic constituents 
of protoplasm renders easy their conversion into different 
compounds; the chains are broken up and when the links 
are reformed they show a different relation to each other. 
The proteins have the greatest lability in this respect. 
From the 21 amino acids which compose them it is theoreti- 
cally possible to derive millions of different kinds of proteins . 

This is one reason why the proteins are regarded as 
responsible for specificity; all species now in the world can 
doubtless be distinguished from each other on the basis of 
the chemical constitution of their proteins. But it should 
be borne in mind that protoplasm never exists entirely 
free of carbohydrates and lipins; that from sugars plants 
make protein; that sugar as a pentose or as hexose is an 
essential constituent of nucleic acid, that lipins are the 
greatest source of energy and are the great binding sub- 
stance; that lipins if not present as such in the nucleus 
probably give rise to the phosphatized portion of the 
chemical substance, nucleo-protein, found in the nucleus; 
and finally, that much of the protein in cells does not exist 
as simple protein but as a conjugant with lipins or lipin- 
derived phosphate. Protoplasm is not a compound but a 
complex of compounds of these three organic constituents 
together with other constituents, water, gases, salts. 

Water is the most abundant compound in protoplasm. 
Roughly, two-thirds of an animal's body is water; in some 
cases it may amount to more than 90 per cent, of the body 
weight. Even water-poor cells, as the enamel of the teeth 
and spermatozoa, still possess a great deal of water. Active 
protoplasm both liberates and utilizes C0 2 and 2 . Dis- 

1 See Bradley and others. 



solved in the water are several inorganic salts, chiefly 
chlorides, carbonates, phosphates of sodium, potassium, 
calcium, etc. 

The researches on the analysis of carbohydrates and on 
the synthesis of sugar, on the analysis of proteins (espe- 
cially nucleo-proteins) stand in the fore-front of bio- 
chemical achievements. And yet, there remains much to 
be learned of the chemical make-up of the cell. Farther 
and more refined analysis may furnish a clue to a better 
understanding of living matter as a complex of compounds. 
Up to now chemical analysis tells us only what is found in 
the living thing after it is killed. The very nature of 
chemical analysis demands isolation of the protoplasmic 
components as separate chemical entities and thereby sets 
up an obstacle in the search for the chemical constitution 
of the living state. To know even with absolute exactness 
the chemistry of each compound in the once living cell does 
not guarantee that we know how these are organized in life. 
Moreover, living organization is dynamic whereas the 
application of chemical analysis by necessity demands 
destruction of the very space-time structure which is the 
changing organization characteristic of life. No single 
datum now at hand warrants the assumption that the 
chemistry of the cell in the chemist's test tube is that of the 
living cell. Indeed, the evidence points to the contrary. 

These shortcomings of chemical analysis of the living 
thing have served to focus attention upon its physical 
attributes. Many biologists, therefore, seek to determine 
the physical diagnostics of non-living and living on the 
one hand, and of the living before and after it is killed, 
on the other. 

The presence of water in large amount confers upon 
protoplasm certain physical properties ; similarly, the 
remaining substances entering into the composition of the 
cell are responsible for other such properties. Recently, 



a great deal of attention has been given to the physical 
properties of the protoplasmic system. A new and flourish- 
ing branch of biology, bio-physics, stresses this aspect 
of the study of life. Some of the more general of these 
physical properties may be mentioned. 

Protoplasm is a liquid capable of flow. This fluidity 
can be demonstrated in several ways. First, direct 
observation shows that in many cells the cytoplasm 
streams. 1 In Amoebae, in many egg-cells, etc., currents 
can be discerned. Cells which fail to reveal the presence of 
such currents often show the cytoplasmic granules in intense 
Brownian movement. Again the fact that in many cells it 
is possible by very slight centrifugal force to shift and to 
separate the cytoplasmic inclusions according to their 
specific weights, demonstrates the liquid nature of proto- 
plasm. Also, one may compress eggs, draw them out into 
long strands or otherwise treat them as if they were elastic 
bags of water. 2 

The cell-liquid and the various formed bodies suspended 
in it reveal that protoplasm is both a solution and a suspen- 
sion, somewhat like milk, for example, which is a solution 
of water, electrolytes, proteins, sugar, and lipins in which 
globules are suspended. Protoplasm being largely made 
up of protein and complex carbohydrates in the colloidal 
state, is itself in this state. A colloid (literally glue- 
like 3 ) is matter in the state which can not or can only 
with difficulty pass through an animal membrane; whereas 
crystalloids, like cane sugar and common salt, can very 
readily pass through such membranes. Protoplasm exhib- 

1 Cf. Goette, 1875, w ^ 10 not 01l h ver y clearly observed proto- 
plasmic streaming in an egg but also proffered an interesting 
hypothesis as to its significance. 

2 Just, 1928a. 

3 Armstrong^ 1927, p. 656, points out the bad usage of the term, 



its many properties characteristic of this state of matter. 
This is not to say that the colloid state is peculiar to organic 
compounds, such as proteins and carbohydrates; the most 
thoroughly investigated colloids are those of inorganic 
substances. It is to be emphasized that the term, colloid, 
applies to a state or condition only and not to a kind of 
matter different from crystalloid. Since we know that 
protoplasm is in the colloid state, it should not astonish 
us that it behaves as other matter in this state. 1 

The specific gravity of the total of the cell-contents is 
usually greater than that of water. But where much fat 
is present, as in some fish-eggs, the specific gravity of the 
cell is lower than that of sea-Water, as shown by the fact 
that such eggs are found floating on the surface of the sea. 
In some eggs the specific gravity increases after fertiliza- 
tion, a change undoubtedly due to loss of water. 

The refractive index of a cell is a physical property 
deserving attention. It depends upon the ground-sub- 
stance and the cytoplasmic inclusions. With the state of 
aggregation of these latter, the shape and the water-content 
of the cell, the refractive index of the cell varies. The 
influence of these factors is shown during the process of 
cell-division when the cell's refractive index changes with 
the dispersion of the inclusions, loss of water and change of 
form of the cell. 

Another physical property of the protoplasmic system 
much studied in recent years is its viscosity. The signifi- 
cance attached to such studies has, in my judgment, been 
grossly exaggerated. Nothing up to now indicates that 
viscosity has the importance for the state of being alive 
assigned it by the various investigators who, with different 
methods, and even with the same method, obtain widely 
diverging results and announce conflicting opinions. The 

1 See Svedberg, 1925, and Heilbrunn, 1928. 



problem of protoplasmic viscosity, with special reference to 
animal eggs, can be dismissed in a few words. 

The statement made above that the cell contains a high 
percentage of water, docs not imply that the cell-contents 
flow as freely as water or that they always maintain the 
same degree of flow. Before the present vogue of measur- 
ing the viscosity of protoplasm it was shown that the cell- 
contents have a higher viscosity than water and that this 
viscosity varies from time to time in a particular cell and 
differs in different cells. One thinks at once of correlating 
viscosity with the water-content of a cell: the more water, 
the less viscosity and vice-versa. To an extent this is true, 
but the situation is not so simple. Take the case of eggs 
actively going through the process of division. During 
each cleavage-cycle, substances move back and forth 
between nucleus and cytoplasm, between cytoplasm and 
cytoplasmic inclusions and between egg and external 
medium. Only in the case of the last named exchange, 
obviously, does the cell lose water; in the first and second, 
water moves from place to place within the protoplasmic 
system. Now since, as the older observations have 
abundantly shown, changes in viscosity parallel the cell's 
rhythmical activity in division, it becomes necessary in 
measuring viscosity-changes to appreciate the fact that 
water does not shift between nucleus (and structures 
associated with it) and cytoplasm only; one must also 
recognize the shift between cytoplasmic inclusions and 
cytoplasm, especially if one estimates the change in vis- 
cosity on the basis of the movement of these inclusions, 
for example, by means of centrifugal force. Since yolk- 
spheres undergo physical changes during cell-division, one 
can not in measurements of protoplasmic viscosity assume 
them as unchanging in deriving conclusions concerning the 
viscosity of the cytoplasm. Also, in experimentally treated 
eggs, one can only draw conclusions as to the effect of the 



means if one knows definitely that this is on the cytoplasm 
alone, or on one or another of the inclusions or on the whole 
of the cell-contents. Finally, the effect of experimental 
treatment should be known to be reversible, and coagula- 
tive death-changes should not be confused with what 
is denominated as the normal viscosity-changes in normal 
and viable cells. Until the viscosity studies have been 
refined, changes in this physical property cannot be 
regarded as an infallible sign of the state of being alive. 

A leading characteristic of biological researches of the 
last twenty-five years lies in the emphasis which came to be 
placed upon the investigation of the physical properties of 
protoplasm — an emphasis so great that it has virtually 
created another department in an already heavily depart- 
mentalized science. Everywhere nowadays bio-physics, 
the physical chemistry of the cell, and the colloid chemistry 
of protoplasm are in evidence. Many an interesting new 
datum concerning electrical conductivity, hydrogen-ion- 
concentration, temperature-coefficients, etc., has been 
accumulated, whilst the older conceptions of osmotic pres- 
sure, surface tension and the like have been refined by 
more exact mathematical treatment. If from all these 
studies far less has come than had been hoped, one benefit 
of them deserves emphasis. No matter how refined a study 
on a physical property of the living substance may be, its 
value for the understanding of vital activities is determined 
by its relation to these vital activities and by its correlation 
with known form-changes and normal processes in viable 
cells. Modern studies in bio-physics and the like have 
served to indicate anew how necessary is adequate knowl- 
edge of the visible structure and the form-changes of cells, 
and how much we still need to extend this knowledge. 
What is called the morphology of a cell — its visible form 
and its visible form-changes — still remains the basis of 
biological investigation both for its own sake and as the 

4 6 


basis of an attack by bio-physicists, bio-colloid-chemists, 
bio-physical chemists, by which may possibly come an 
elucidation of the problem of vital manifestations. I 
therefore turn to a description of the cell. 

Under low power of the microscope, a living unfertilized 
egg of the sea-worm, Platy nereis, mounted in a few drops 
of sea-water, at first glance appears as a greenish-yellow 
sphere everywhere crowded with smaller spheres except 
near the centre and at the periphery beneath the well 
marked egg-membrane; the whole presents a pebbled or 
shagreen effect. A second glance, especially if the egg is 
brought into focus so that one obtains an optical section 
of it, gives clearly the regions first noted. The large clear 
area near the center is the nucleus, sharply set off from 
the remainder of the cell by its transparency and apparent 
homogeneity. Outside of it, closely crowded against the 
nuclear boundary are innumerable spherules with some 
twenty or more refringent globules (oil drops) interspersed. 
Among these spherules and globules minute bright granules 
are scattered. Under higher magnification still smaller 
granules may be discerned. Spherules, globules and 
granules lie in the endoplasm or inner cytoplasmic region. 
Beyond it is a clear band, the ectoplasm, or outer region of 
the cytoplasm. In this egg the ectoplasm is crossed by 
fine radial lines; these appear to reach the egg-membrane, 
called the vitelline membrane, though actually they end 
in the plasma-membrane which is difficult to discern and 
which lies underneath the vitelline membrane. 

If we fix this egg quickly with some chemical reagent 
which preserves it in a state closely resembling the living, 
and cut it up into thin slices, we easily discern structures 
seen with more difficulty in the living cell. Figure I is 
of a stained slice from such a fixed egg. In the nucleus, 
granules of various sizes and two chromosomes are shown. 
These bodies lie in a faintly granular grayish-blue field 



enclosed by the nuclear membrane. The cytoplasm is 
sharply differentiated into two regions; the endoplasm, 
crowded with yolk spheres, oil drops and granules, the 
smaller of which are mitochondria; and the ectoplasm with 
its radially disposed lines which extend to the plasma- 

« - 






p . 

Fig. i. — Section of an unfertilized egg of Platy nereis megalops. Surrounding 
the large nucleus, germinal vesicle, is the cytoplasm, which is clearly marked off 
into two zones, the endoplasm and the ectoplasm. 

membrane beneath the vitelline membrane. The egg of 
Arbacia, a sea urchin, is similar to that of Platynereis 
except that the whole egg including the nucleus and the 
endoplasmic bodies is smaller, the ectoplasm a mere line, 
and the vitelline membrane an extremelv fine film. In 



all cells nucleo-cytoplasmic organization is visible and 
ecto-endoplasmic differentiation is expressed. 

Nuclei may vary in size, but their size is generally 
constant for a given species of cell, though this varies during 
the process by which the cell reduplicates itself. In eggs 
it also varies during the end-stages of ripening, as will be 
shown later. 

The nucleus also shows diversity in form. Nuclei though 
most frequently spherical or oval or tending toward these 
two forms, may be quite irregular as those found in white 
cells of human blood, for example. The amoeboid, irreg- 
ularly form-changing nuclei in the spinning gland cells of 
butterflies have often been described. Again, during the 
process of division an otherwise spherical or oval nucleus 
may be very irregular due to incomplete or slow fusion of 
the separate chromosomes into one nucleus. This is 
clearly shown in nuclei of some eggs, as those of the thread 
worm, Ascaris, the water-flea, Cyclops. In these the 
nucleus may be regarded as a composite of many smaller 
nuclei, each containing one chromosome. Although this 
condition is not met with in all animal cells, it may 
be regarded as a fundamental condition of nuclear 

That the chromosomes are the best known structures 
in the cell is due in part to the fact that they can be so 
easily studied because of their affinity for colors, which 
gave them their name. In the living cell 1 they often appear 
as refringent drops of the same shape and approximately 
the same size as when fixed and artificially stained with one 
or another dye. A constant number of chromosomes is 
characteristic for the cells of a given species of animal. 
This number is best ascertained in fixed and stained cells, 
though it can be made out in some living cells, while they 

1 Flemming, iSyg^ Peremeschko, 1879. 



are in stages of nuclear division for then the chromosomes 
are most easily visible. 

The chromosomes also appear as strands of condensed 
structures lying in the more fluid substance of the nucleus, 
the so-called karyolymph. When the nucleus is at rest the 
chromosome-material appears as granules and is designated 
chromatin. How far this appearance coincides with the 
real state of the chromosome-material has yet to be deter- 
mined. Since the chromosomes display regularity in struc- 
ture and behavior during the process of nuclear division 
when they are visible and, after fading from view, appear 
again as bodies of the same number, form and character, it 
lies close at hand to conceive that they maintain their 
organization also when they are seen as granules. Theo- 
retically, for the chromosome-theory of heredity, which will 
be discussed later, it is also simpler to conceive that the 
chromosomes always maintain their form and never 
become disorganized by breaking up into granules. 

The rapidity with which the nucleus carries out its 
changes might be considered sufficient for the maintenance 
of integrity during both the resting and the dividing stage. 
But the fact that the nucleus remains separated from the 
cytoplasm may be due to an interposed membrane. 
Though such a membrane can be demonstrated in many 
cells, its presence generally in cells has been denied. It is 
further not at all clear how this membrane arises. As with 
the presence or absence of the nuclear membrane, so with 
the linin, a net-work of material in the fixed nucleus: some 
hold that it is a real structure, others that it is merely an 

There is present in the nucleus a nucleolus (in some cases 
there are several nucleoli) which may be a double structure. 
Its function is not known. Indeed, one can not be sure of 
its origin or fully follow its history from one cell generation 
to the next. 



The chemistry of the nucleus is known from studies made 
especially on blood cells and spermatozoa in which the 
nuclear material is rich in comparison with the cytoplasmic. 
Since in the spermatozoa the nuclei (sperm heads) are 
chromatin in the most condensed state known, their 
-chemisty is practically the chemistry of chromatin. The 
following is the chemical constitution of an animal's cell- 
nucleus (spermhead of the white fish) : 

C96.Hi84-N 5 4.O22(C43H57Ni 5 P4O30)4 ~ 24H2O 

The nucleus essentially is made up of a protein conjugated 
with a sugar-containing compound which through the 
presence of phosphoric acid becomes an acid, nucleic acid. 
The presence of phosphorus suggests that although lipin 
may not be present as such in the nucleus, it is necessary for 
the formation of nucleic acid. Nucleo-protein contains 
protein and carbohydrates which probably come together 
through the mediation of phosphorus-containing lipin. 
In the light of this suggestion the synthesis of nucleo- 
protein represents the highest chemical activity of the 
living substance since we see here synthesized the three 
important substances that distinguish living from non- 
living matter chemically: protein, carbohydrates and fats. 
For this reason I consider it an approach to the understand- 
ing of chemical activities in living matter to study nucleo- 
protein (and especially nuclein) with respect to its rate and 
duration of formation and its amount. 

In dividing cells the intact or so-called resting nucleus 
becomes active and exhibits changes after which it divides. 
However, the nucleus is not to be thought of as a body 
implanted in the cytoplasm with no relation to it except 
during synchronous division. Since in the so-called resting 
stage the nucleus continually increases in size to the 
moment in which the activities leading to its division set in 
we assume that it takes up material from the cytoplasm — 



substances in solution. Indeed, with respect to the nucleo- 
cytoplasmic relations, the so-called resting stage of the 
nucleus is as important as the stage of division. 

During the stages of nuclear division there also occur 
changes in the cytoplasm, as changes in refraction, in 
dispersion of granules, changes within the granules them- 
selves. The fact that these changes can be correlated 
with the stages of nuclear behavior suggests that chemical 
syntheses and analyses in the living cell are in part at 
least controlled by the movement of substances in solu- 
tion to and from the cytoplasm out of and into the nucleus. 
To this point I return later. 

As has been said above, the cytoplasm is composed of an 
inner core immediately surrounding the nucleus and, 
external to this, an ectoplasmic region extending to the 
cell-membrane. In this thus differentiated area of extra- 
nuclear protoplasm formed bodies of different size are 
suspended. By determining what in this extra-nuclear 
mixture of fluid and suspended bodies is indispensable to 
the life of the cell, we may derive a definition of cytoplasm. 

The suspended bodies are chiefly: droplets of oil, yolk, 
small formations known as Golgi-bodies, granules called 
mitochondria and chromidia; further, starch, crystals of 
various kinds, secretion-granules, excretory products. But 
these cytoplasmic inclusions are not to be taken for the 
cytoplasm itself, as is strongly indicated by the following 
facts. First, animal cells vary with respect to the content 
of cytoplasmic inclusions: not all cells contain them all and 
some even under high power of the microscope appear to 
contain none. Second, in many cases the inclusions occupy 
no constant position in cells: thus yolk generally present in 
eggs is variously distributed. Third, and most important, 
portions of eggs devoid of all cytoplasmic inclusions having 
been fertilized develop as do whole eggs with all inclusions 
present. Consider the egg of the marine worm, Chaetop- 


tents, after it has been centrifuged with force sufficient to 
stratify its inclusions according to the specific gravity of 
each: between the zone of oil-drops, the lightest constituents 
of the cytoplasm, and the yolk-spheres massed at the 
opposite pole, lies a clear zone which under high power of the 
microscope and with bright-field illumination appears 
optically empty; with the dark-field or after proper fixation 
this zone appears to be made up of extremely fine granules. 
This clear area if cut away, even though it be but a small 
fraction of the egg, will develop if fertilized with almost the 
same pattern and tempo of development as the whole egg. 
This is likewise true of eggs of sea-urchins. The conclusion 
is that of the extra-nuclear substances the only part abso- 
lutely necessary for life is the clear inclusion-free men- 
struum. I therefore define as cytoplasm this menstruum 
only. In the following pages I shall adhere strictly to this 

Despite the fact that the cytoplasmic inclusions are not 
essential to cytoplasm, they have undoubted functions and 
merit here brief notice. In the following treatment, I speak 
mostly of egg-cells. 

The lipins (fats and fatty substances), discussed above, 
are present in eggs largely as oil and in yolk (lecithin). 
The oil (fluid fat) in eggs exists as drops in various degrees 
of emulsification. Whilst it is easy to observe oil drops in 
some eggs in which they are relatively large — as in 
the egg of the sea-bass, — it is often difficult to observe them 
in some other eggs where they are in a state of fine division. 
In the unfertilized egg of Nereis one can count the twenty to 
twenty-two oil drops present and can as development 
ensues after fertilization follow the gradual reduction by 
coalescence of this number to four. In the living larval 
worm one can further watch the emulsification of these into 
finer and finer drops which eventually disappear — as further 
shown by micro-chemical tests on both living and killed 



larvae. This observation teaches us one function of the oil 
in this egg-cell: it is a reserve food-material utilized only in 
the period before the young worm actively begins to ingest 
food. A gram of fat, as is well known, is richer in energy 
than a gram of carbohydrate or of protein. 

Lipin plays a role both in the water-holding power of 
cells and in the cells' immiscibility with the surrounding 
medium. It exists in the cell in other forms than oil drops. 
So, cholesterol is said to be found in the membranes of some 
eggs; the human red blood corpuscle likewise contains it. 
A closely related compound, ergosterol, is nowadays much 
discussed because of its significance in vitamin-chemistry. 
Fat is also associated with pigments and thus may play a 
part in oxidation-processes. Yolk is another form in which 
lipin occurs in egg-cells. 

Yolk in eggs appears in manifold chemical and physical 
forms. The identification of yolk-bodies in eggs rests 
largely upon staining reactions. Spherules in the cyto- 
plasm which are stained dark blue or black with iron 
haematoxylin are usually assumed to be yolk. If finally 
they come to lie in the endoderm cells one concludes that 
they are food material and presumably yolk. Even so, 
these spherules show striking differences. Take, for 
example, the yolk spheres in the eggs of two closely related 
marine worms, Nereis and Platynereis. In the former egg 
the yolk spheres are homogeneously blackened by haema- 
toxylin; in the latter every yolk sphere is a delicately 
reticular structure staining faintly with the haematoxylin. 
In each case these bodies come to lie in the four cells which 
compose the gut. 

At the beginning of the process of incubation, yolk 
constitutes the greatest bulk of the chick's egg^ whereas at 
the time of hatching the yolk has largely disappeared. 
This fact means, as we shall see in a later chapter, that yolk 
has been converted into cytoplasmic substance. In the 



same way, of course, the growing animal after having been 
hatched from the egg manufactures its peculiar kind of 
protoplasm from the food which it takes in. So also yolk 
is used up in the development of other large eggs, as those of 
fishes and amphibians; in smaller eggs, however, develop- 
ment proceeds to the stage of hatching without the utiliza- 
tion of yolk. For example: in the Nereis egg, yolk is not 
used during cleavage by the blastomeres; it goes into the 
four macromeres which form the gut. A sea-urchin egg, in 
which the yolk has been displaced by centrifugal force, 
develops perfectly. And yolk-free fragments of marine 
eggs develop normally as stated above. 

In the egg of Nereis the yolk spheres are polyphasic, i.e., 
they contain a protein-skein and lipin. If these eggs are 
placed in hypotonic sea-water, oil in minute drops escapes 
from the yolk-spheres, leaving a reticular and more watery 
medium, the whole enclosed by a strong membrane. The 
yolk spheres now resemble those present normally in the egg 
of Platynereis. On transfer to normal sea-water the oil 
re-enters the yolk-spheres and their original optical proper- 
ties are restored. 1 The process is thus reversible. An 
observation like this shows how yolk may vary and that 
we can not satisfy ourselves with only a rough identification 
of it as that by staining. Certainly, what thus we 
identify as yolk is not a pure lipin. 

The Golgi-bodies or Golgi-apparatus, commonly a net of 
fibres or a cluster of granules of varying form and size, 
have been described for almost every type of animal and 
plant cell. Their meaning and function are yet to be 
made clear. Undoubtedly in some cases what have been 
described as Golgi-bodies are drops of fat; 2 in other cases 
even vacuoles have been described as Golgi-apparatus. 

Just, 1926. 
Just, 1927a, 



Mitochondria are minute spheres, rods or filaments. In 
the egg of Platy nereis pictured they are rods; while in that 
of the closely related Nereis they are spheres. In some 
cells they assume first one then the other form. It has 
been held that the mitochondria are phospholipid. How- 
ever, because with the most careful cytological technique 
yet devised (that invented by Altmann), later workers 1 
were unable to prove that mitochondria contain fats but, 
instead, could demonstrate their protein nature, doubt is 
thrown on the older view. Certainly in those marine eggs 
which I have studied on this point, the mitochodria do not 
reveal the specific gravity of fat; for, when the eggs are 
centrifuged, instead of lying in the zone next to the fat 
they take the place above the yolk. 2 

Chromidia are generally believed to be cytoplasmic 
granules derived from the nucleus. I may refer again to 
the egg of Nereis: in it one can observe granules which 
seem to come out of the nucleus, wander into the cytoplasm 
and come to lie at the cell-periphery in which position 
they can not be distinguished from mitochondria. Under 
various experimental conditions, as after ultra-violet radi- 
ation or by treatment with hypotonic sea-water, the 
number of such granules in the vicinity of the nucleus is 
increased. Often also with such experimental means chro- 
mosomes are eliminated which degenerate into granules 
not easily, if at all, distinguishable from chromidia and 
mitochondria. This degeneration of chromosomes is quite 
different from the degeneration of chromosomes in abnormal 
eggs which fail to develop; these latter can never be mis- 
taken for chromidia or mitochondria because structurally 
they offer an entirely different aspect. On the basis of 

1 Bensley and Gersh, fQJJ- 

2 Bensley and Gersh are extremely cautious in their conclusions 
and it may be that even under the conditions of their experiments the 
lipin-fr action of the mitochondria zvas d est roved. 



my observations 1 venture the opinion that chromidia and 
mitochondria represent stages in the transformation of 
granules of nuclear origin. 

Of granules other than mitochondria and chromidia 
found in eggs, pigment is most striking. Many eggs seen 
en masse have beautiful colors — various shades of red, 
orange, yellow or green. There are some species which 
have eggs of two distinct hues; thus individuals of the 
little marine worm, Aiitolytus varians, have either red or 
green eggs. The pigment of sea-urchin eggs has been 
shown to be a lipochrome. 

Occasionally crystals can be demonstrated in eggs. Thus 
Aleves has described fine slender bodies found in fertilized 
eggs of Psammechinus and I have found them also in ferti- 
lized eggs of Echinarachnius. That such formations are 
increased in cross-fertilized eggs 1 I do not doubt, but that 
they are solely due to the effect of the foreign sperma- 
tozoon is to be questioned in view of Aleves' and my own 

Some of these cytoplasmic inclusions, as well as others, 
that are not here discussed as, for , example, secretion- 
granules which leave the cell, are certainly temporary 
structures. Study of the history of the egg from its 
earliest formation, when its cytoplasm always appears 
homogeneous, through the stages during which it becomes 
laden with inclusions, teaches that inclusions appear in the 
cytoplasm during development. The building up of cyto- 
plasmic inclusions is an indication that the egg takes in 
food-material from its surrounding medium. Indeed, in 
some eggs, as those of the flatworms, the yolk, elaborated 
by special gland-cells, is merely deposited around the egg. 

In limiting the application of the term, cytoplasm, to 
the clear and almost homogeneous menstruum or ground- 

1 Tennent, IQ20; Hibbard, IQ22. 



substance and thereby dismissing the various inclusions 
suspended in it as necessary constitutents of cytoplasmic 
structure, we dismiss much of the work done on the chem- 
istry of the cell as relevant to the chemistry of the cyto- 
plasm inasmuch as chemical studies have been made so 
largely en gros — encompassing everything including food- 
material and effete matter which might be present in each 
of the thousands of cells which are often necessary for a 
single chemical determination. The chemistry of the cyto- 
plasm, from my point of view, would embrace only the 
chemistry of the inclusion-free ground-substance. Since we 
do not yet possess such, we can only approximate it by 
considering that which remains after we have subtracted 
from what is known of the chemistry of the whole cell that 
which is known of the chemistry of the nucleus, of the 
various inclusions and of the cell-membrane. This so little 
light does not throw its beams far and we remain still much 
in the dark. Moreover, there is always the obstacle that 
what we call the chemistry of the whole cell is not that of 
living protoplasm; the isolation necessary for chemical 
study may change the cell-constituents. Micro-chemical 
studies on cell-inclusions are certainly valuable as are those 
on the inclusion-laden protoplasm. But there is the great- 
est need for such studies on the ground-substance itself. 

The definition of cytoplasm here given makes mandatory 
a revaluation also of studies on the physical properties of 
protoplasm. Here, in order to know what properties inhere 
in the ground-substance, unless this be studied as such, the 
known properties of nucleus and cytoplasmic inclusions 
should be subtracted from those known for the whole cell. 
It is a curious fact that in that domain of physical chemis- 
try which by its definition we should expect to encompass 
the study of the ground-substance workers have laid more 
stress on the whole cytoplasmic area and neglected the 
possibility of investigation of the ground-substance which 



when isolated from, and free of, cytoplasmic inclusions is 
wholly viable. I refer to that branch of chemistry, colloid 
chemistry, so-called, where fact and fancy are dispersed in 
the medium of a naivete which so often characterizes new 
adventures along narrow confines. 

An egg like that of Nereis in which the formed bodies, 
oil-drops and yolk spheres, are visible when magnified only 
twenty times can not with respect to these bodies be con- 
sidered by a colloid chemist as other than an extremely 
coarse suspension. It can not be regarded as a colloidal 
solution for a colloidal solution is usually defined as one in 
which the particles range in size from I, 2 or 6 to 250 ma. 
The smallest are at the lowest limits of visibility with the 
ultra-microscope and the largest come just within range of 
microscopic observation. Even such eggs as sea-urchins', 
whose oil-drops, yolk-spheres and pigment granules are 
smaller than the oil-drops and yolk-spheres of the Nereis 
egg, can not be looked upon as colloidal solution for their 
inclusions are visible under relatively low power of the 
microscope and thus do not fall within the range of sizes of 
particles in a colloidal solution. But the ground-substance 
of these eggs is a colloidal solution since its particles, barely 
visible under the highest powers of the microscope, are 
more clearly revealed by dark-field illumination. 

The definition of cytoplasm here given also means dis- 
carding theories of the morphological structure of cytoplasm 
since these, the alveolar, filar and granular theories, refer 
to the cytoplasm plus inclusions. 

Even if we would define cytoplasm to embrace the cyto- 
plasmic inclusions, the alveolar theory of cytoplasmic 
structure would be untenable. In the first place, although 
it is true that there are cells which show a vacuolated or 
foam structure, this is by no means all-pervading but is 
limited to certain cellular regions. Moreover, such struc- 
ture is far from being demonstrable in all cells and the 



experimental evidence indicates that it is non-essential to 
the living cell-substance. Secondly, the alveolar structure 

Fig. 2. — -Two sections of the unfertilized egg of Nereis limbaia (after Lillie). 
a is badly fixed as shown by the partially dissolved yolk spheres and the wholly 
dissolved oil drops which in b are intact. Compare b with the figure of the living 
Qgg as shown in Fig. 22 (p. 158). v.m., vitelline membrane; c.l., ectoplasm. 

described for fixed cells, as it often has been, is the result 
of improper fixation. In such cells the cytoplasm appears 
a mesh, empty holes occupying the greatest space, holes 

60 * 


which have been brought about through the action of the 
fixing solution In dissolving out fat, yolk or other cyto- 
plasmic inclusions. Compare the pictures given, (Fig. 22) 
of a living, (Fig. 2a) of a poorly fixed, and (Fig. lb) of a 
properly fixed egg. In Figs. 22 and 2b oil-drop and yolk- 
sphere are intact. When one bears in mind that Fig. 2b 
is from a very thin section, about one-thirtieth of the entire 
egg, and that the larger inclusions present can very easily 
be displaced in sectioning, the picture is all the more con- 
vincing: the resemblance to Fig. 22 is striking. But 
compare Fig. 2a — a mesh-work like a fish-net. From 
pictures inferior to this (Fig. 2a), wholly disregarding the 
structure seen in the living egg, Wilson derived his support 
of the alveolar theory of protoplasmic structure. 1 When 
one speaks of the physical basis of life and refers only to 
the morphology of the cell, one is misleading 2 — doubly so 
if one has in mind a dead cell showing mostly holes. True, 
protoplasm is the physical, i.e., the corporeal basis of life; 
also in the fixed state this basis is of interest. But we shall 
not understand it, if first we vacuolate it and then ascribe 
to the holes essential meaning without knowledge even as 
to the significance of the substances which we replaced by 
holes. On the other hand, to say that the cytoplasm of 
the living cell is alveolar in structure because the holes 
of the dead cell were occupied by formed bodies is to give 
new meaning to the term, alveolus; I can not see how a 
droplet of fat or a spherical yolk-mass can be designated 
an alveolus. I have often pressed them out of an egg into 
sea-water where they remain intact. If they are moved by 
centrifugal force from one to another region of the cell, 
they do not leave empty spaces behind them. Hence, 
neither they nor the places they occupy in the fluid cyto- 
plasm are alveoli. 

1 Wilson, 1926; but cf. Mathews, f<?o6. 
- Wilson, I.e. 



But let us for the moment dismiss badly fixed eggs, 
pictures of fenestrated or lacunar substance, also the empty 
spaces previously occupied by oil and yolk, and extend the 
meaning of the word alveoli to embrace such inclusions as 
oil-drops and yolk-bodies seen in the living egg as discrete 
spheres. How far does this assumption strengthen the 
alveolar theory of protoplasmic structure? Can we con- 
clude that inasmuch as each " alveolus" (oil-drop or yolk- 
sphere) is laid down by the egg during its history, an egg in 
that stage before oil or yolk is deposited as easily visible 
discrete spheres has no structure? Shall we say that in 
those eggs which distribute their "alveoli" in such manner 
that some cells possess all the "alveoli" and others none, 
those cells which do not contain them are structureless? 

The alveolar theory has found support from the fact that 
certain artificial mixtures resemble some kinds of proto- 
plasm. Witness emulsified oil. If we examine under the 
microscope a thin layer of mayonnaise — olive oil and 
vinegar plus egg-yolk, the emulsifying agent — we find that 
the oil is in drops and among them are scattered yolk- 
spheres. By carrying the process of emulsification further 
and further, we obtain smaller and smaller oil-drops and in 
this way make structures that resemble those of eggs with 
larger or smaller oil-drops. Such a film of mayonnaise 
when fixed by a proper agent for cytoplasmic fixation 
retains faithfully its appearance before fixation and 
simulates the picture of properly fixed egg-cytoplasm. The 
emulsification obtained by beating olive oil with vinegar or 
lemon-juice, without egg-yolk, though less permanent, 
bears also a close resemblance to the living cytoplasm of a 
living egg, say that of a star-fish. It has even been held 
that protoplasm is a foam-structure because of the resem- 
blance of some cellular structures to such foams prepared in 
the laboratory. As I see it, all that these and similar 
models tell is that the living cell can hold its oil in the form 



of discrete drops of larger or smaller dimensions. They 
give us no information concerning the mode in which the 
living cytoplasm moulds the oil-drops — now coalescing 
them, now emulsifying them. They fail to reveal the value 
of such structure for life phenomena. Finally, for the 
alveolar theory, a yolk-sphere is as much an " alveolus" as 
an oil-drop; and yet although we do not know as much 
about yolk as we would wish, nevertheless, what knowledge 
we have is sufficient to warrant the conclusion that both 
physically and chemically a yolk-sphere is something quite 
different from an oil-drop; a yolk-sphere, unlike an oil-drop, 
is a combination of fat and protein, 1 If one adheres to the 
alveolar theory by denominating oil-drops and yolk-spheres 
" alveoli," one excludes from consideration the differences 
between these " alveoli," thus stressing their shape rather 
than their physico-chemical make-up. 

The filar theory of cytoplasmic structure never has 
attracted many adherents, doubtless because of the limited 
occurrence of fibrils in cytoplasm. For the most part, when 
found, they are in fixed cells, in which, before fixation, they 
can not be seen. 2 The evidence for their presence in some 
other living cells is by no means convincing. Thus, the 
cytoplasm of outward flowing cell-processes appears to be 
filar — but this may be due to granules disposed in rows by 
the streaming of the medium that is forced through the 
orifice. 3 Mitochondria-granules may by close alignment 
appear as fibrils. 

Altmann 4 suggested that granules in the cytoplasm are 
the elementary living substance. Both before and since 
his time others have put forth similar theories, often includ- 
ing more kinds of cellular granules than Altmann did. 

1 See also Konopacki, 1929. 

2 Lewis and Lewis, 1924. 

3 See Butschli, 1S90; also Just, iQ2Se. 

4 Altmann^ 1890. 



Undoubtedly, Altmann's granules are mitochondria; as 
such they are only cell-inclusions. As to other granules 
described in this connection, one must remember that many 
granules in cells are purely secretory — that is, precursors of 
the cell's secretion, as the granules found in a salivary gland 
or in the pancreas — that some others are storage-stuffs, 
others, excretory products. In other words, granules are 
derivatives of cytoplasmic substances or expressions of 
cytoplasmic activity. That to which they owe their origin 
and not they themselves would seem more likely to be basic. 

For the reasons given, I think that we may discard the 
alveolar, filar and granular theories of cytoplasmic struc- 
ture. Oil-drops, yolk-spheres, threads and granules in the 
cytoplasm make the contents of a cell a gross suspension. 
Such a suspension as that of an egg of Nereis or of a sea- 
urchin does not represent the basic structure of life and 
its peculiar physical state does not offer any clue to the 
solution of the problem of vital activity. 

Turning to the menstruum containing the formed bodies, 
the ground-substance, we find, as stated above, that it 
appears to be almost optically empty. If, however, one 
examines the hyaline region of a centrifuged egg of Arbacia, 
Nereis or Chaetopterus, with extreme care without pressure 
on the egg — for pressure induces abnormal formations — one 
can observe very fine granules as barely visible points. 
Thus, as also said above, the ground-substance is to be 
considered as a colloidal solution, for it has particles below 
the size seen under the ordinary microscope, but which can 
be seen under dark-field illumination. Until we know more 
of the physics and chemistry of the ground-substance, we 
must derive a conception of its structure by observing it in 
the living state and when properly fixed. The fact that 
well-fixed ground-substance so closely resembles the living 
makes fixation a supplementary aid in its investigation and 
permits us to draw conclusions concerning the living state 

6 4 


from the fixed. Processes too fleeting to be followed 
exactly in the living can be followed in their single steps in 
the fixed. By thus supplementing the study of the living 
by that of the fixed ground-substance, we may approach 
the understanding of the basic structure of cytoplasm. My 
confidence in the value of the study of properly fixed cells 
rests upon the fact that such study has confirmed my 
observations of the extremely delicate momentary changes 
in the ectoplasm of living cells. Without ever discounting 
the postulate that the living cell is the basis, control and 
aim of biological investigation, I have come to appreciate 
the value of proper fixation. But since the use of fixation 
has been and is questioned by many workers, I must for a 
moment dilate on this point. 

When at the end of the last century Fischer 1 and Hardy 2 
independently inveighed against fixation of protoplasm, 
neither made much impression. But the impression grew; 
to-day we discern a reaction of no mean proportion against 
the study of fixed cells. Some histologists adopt an 
apologetic air, offering their findings on fixed tissues in all 
but a furtive manner; others have given themselves over 
to the cinematograph or to the culture of living tissue 
removed from the animal. 

Now without doubt conclusions as to living protoplasmic 
structure drawn from study of dead cells should ever be 
subjected to severest criticism. He who works with dead 
cells should never be under delusions that he has to do with 
other than a dead thing. I do not believe that any com- 
petent histologist takes any other view. And yet, there 
is often inference by those who hold lightly all work on 
fixed cells, whatever its purpose or conclusions, that the 
investigator of fixed, sectioned and stained cells forgets 

1 Fischer^ iSgg. 

2 Hardy, 1S99. 



not only that he deals with dead matter but also that he 
subjects the corpse to some thirty separate treatments 
before he places sections of it under his microscope for 

Hardy's paper nowadays hailed by so many as having 
infallible authority falls short in three directions 1 : First, 
Hardy studied the action of too few fixing solutions; second, 
he failed to make comparisons of the fixed with the living 
cells; and third, he used cells in bad condition. 

Every student of histology knows that he can not assume 
that because he has successfully fixed cells of one tissue he 
can employ this same fixation for every other kind of cell. 
The technique of fixation has not passed very far beyond 
the trial and error stage and, therefore, every new type of 
cell encountered offers a new problem of fixation. Hardy 
should have used more agents and of a much more diversi- 
fied constitution instead of holding so strictly to those of 
closely related composition. 

Hardy made comparisons between the fixed cell (for 
example, gut cells of Onisctis, the so-called pill-bug) 2 and 
protein solutions and came to the conclusion that his fixing 
agents act upon the protoplasm as they do upon protein. 
This conclusion revealed nothing new. It would have been 
far more valuable had he made comparisons between the 
living and the fixed cells in order to ascertain how far was 
the deviation of the fixed from the normal. Further, Hardy 
investigated always cells of tissues and never free-living 
cells as Protozoa or eggs in their normal environment. This 

1 In his more thoroughgoing work Fischer contributed to our 
knowledge of microscopic technique with respect to the use of dyes. 

2 I may point to the fact that the choice of this object, gut cells of 
Oniscus, was most unfortunate, for several reasons. These cells, for 
instance, contain much chiiin whose presence prevents uniformity 
of fixation. See further: Scheikewitsch, fSpj; McMurrich, fS<?5; 
Conklin, iSgy. 



fact warrants my third objection to his conclusions for two 
reasons: First, his cells were already in some state of post 
mortem changes of greater or lesser degree depending upon 
the time elapsing between their removal from the animal 
and their fixation — these changes being more rapid, and, 
therefore, a more serious source of error, for tissue-cells 
removed from the warm-blooded animals which he used. 
In the case of warm-blooded animals it would also make a 
great difference whether or not the animal from which the 
tissues were removed, was anaesthetized. Second, he made 
no allowance for the bulk of tissue used; that is, his con- 
clusions would have been far more sound had he used a thin 
sheet of tissue made up of a layer of one or two cells instead 
of compact masses from the glands investigated. Not only 
were his cells in some degree of post mortem change, but also 
were they fixed unevenly and cut into sections of different 

True, there is bad and unreliable fixation of eggs. Never- 
theless, it is erroneous to condemn all fixation. If I find 
that after fixation an egg very closely resembles the living 
I can draw conclusions on the basis of this resemblance espe- 
cially since knowing that the cell is dead I take into con- 
sideration that its proteins are changed — coagulated, 
gelated, precipitated, and that still other changes have 
taken place. Instead of abandoning the study of fixed 
cells we should use proper fixation in order to check and 
extend observation on the living. Neither the cinemato- 
graph nor tissue culture will quite replace the good fixation 
of the cell for comparison with the living. 

Although our ignorance of the ground-substance is pro- 
found and will remain so until we study it frankly as such, 1 
isolated from all the coarse particles suspended in it, never- 
theless we have at our disposal certain evidence which may 

1 Just, 1936b. 



form the basis for the extension of our knowledge. That 
the clear, nearly homogeneous, almost structureless ground- 
substance of an egg can (with a nucleus) develop, renders it 
at once amenable to all treatment given an entire egg. As 
it develops it exhibits the same form-changes. Here I 
restrict myself to the discussion of one well-known phe- 

Fig. 3. — Boveri's classic figure of a mitotic spindle in the egg of Echinus 
microtuberculatus. But cf. the aster in Fig. 29b (p. 174) and the mitotic complex 
in Fig. 33d (p. 259). 

nomenon, namely, the appearance of the radiating struc- 
tures seen in so many cells during cell-division. 

In many cells division of nucleus and of cytoplasm occur 
simultaneously. Division of the nucleus may be a very 
simple direct process, a constriction and separation of the 
constricted parts or a more complex and indirect process 
with the chromosomes going through a very orderly 
sequence of manoeuvres on a bundle that appears to be 
made up of threads. In this latter type of nuclear behavior 
there often appear in the cytoplasm (occasionally in the 



nucleus) radiations from each polar end of a group of fibre- 
like structures, the so-called mitotic spindle. (Fig. 3.) 
The radiations center at a minute granule or at a larger 
vacuolar sphere-structure; in some cases both granule and 
sphere are present. The minute body is the centriole or cen- 
trosome and the sphere is the centrosphere; the radiations 
because of their disposition are called astral rays. Best 
seen in cells that possess granules, the rays are also demon- 
strable in cells which are granule-free. Further, cells which 
show no rays are often filled with granules. Therefore the 
rays do not depend upon the cell-inclusions. Seen in the 
living egg of Platynereis^ for example, every astral radiation 
is a granule-free track whose greater width is about 3 ju. 
In the living Arbacia or Echinarachnius egg the rays have 
the same configuration. Nothing in the living or properly 
fixed egg appears as the stiff coarse " astral fibres" of poorly 
fixed eggs. The radiations are without doubt no fibres 
at all. It is often held that they are paths of flow and in 
living granule-rich cells they do certainly appear to be such 
-the flow causing a displacement and close packing of 
granules and thereby accentuating the radiate appearance. 
There is no unanimity of opinion concerning the nature 
either of the radiations or of the spindle-" threads" on 
which the chromosomes move. I share with others the 
idea that spindle-" threads ' ; and astral radiations are 
both extremely fine delicately thin sheaths enclosing 
fluid material, that they are not in any sense fibres with 
contractile power. (See Fig. 29b and Fig. 33d.) 

As an almost perfectly homogeneous and fluid system, 
cytoplasm could scarcely show differentiated pathways of 
flow even in extremely thin-walled tubes; less could it show 
contractile fibres. Either then cytoplasm is not structure- 
less or it has power to elaborate structure sufficient to 
account for the ray-configuration of the astral system. In 
either case the cvtoplasm is different before and after the 



appearance of the rays. These rays thus indicate structure 
in the cytoplasm or at least the capacity of the cytoplasm 
to form structures. 

So far I have dealt with the cytoplasm, the ground- 
substance outside of the nucleus. I should remind the 
reader that I consider the nucleus likewise as differentiated 
ground-substance. The formation of intra-nuclear spindles 
just referred to, the origin of centrosomes from nuclei, 
sometimes described, can be interpreted on the same basis 
as the formations in the cytoplasm. Nucleus and nuclear 
constituents are themselves conceived as expressions of the 
capacity of the ground-substance to form structures. 

In its other derivative, the ectoplasm, the ground-sub- 
stance likewise exhibits capacity for structural formations. 
Indeed, ectoplasmic structure in many cells, e.g., Protozoa, 
is a diagnostic characteristic. I wish to consider this 
peripheral ground-substance as it appears in animal cells. 
But first I must discuss the question of the cell-membrane, 
because it has obfuscated a proper appreciation of that part 
of the cell beneath the membrane, the ectoplasm. 

The cell-membrane, like many another cell-structure, is a 
perennial subject for debate. The majority opinion is that 
cells are invested with membranes but the minority is as 
insistent that a membrane is neither present nor necessary. 
Of those in the camp of the majority, some hold that the 
cell-membrane is an endogenous structure, a living thing 
built by the cell; whilst others deny its life, considering it a 
sort of excretion; and still others assert that it is a formation 
induced by the cell's external medium. Opponents of the 
membrane's existence declare that since the cell-substance 
is in the colloid state, it is unnecessary to postulate the 
presence of a membrane: the mere boundary of the colloidal 
system is sufficient to preserve the cell's integrity. Accord- 
ing to them the cell is somewhat like a drop of oil in water; 
just as the surface of the oil keeps the oil intact, so does the 



surface of the more heterogeneous cell-substances prevent 
cellular disintegration. Also, are there those who unwit- 
tingly hold a foot in each camp. 

Now, whenever there are so greatly divergent opinions, 
one suspects either insufficient factual basis, or confusion 
arising from too loose definition. I strongly suspect that 
the difficulty here arises out of the fact that investigators in 
their discussions are often not dealing with the same thing; 
much of the argument then is beside the point. The 
cellulose wall of a plant cell is something quite different 
from the surface of a Paramoecium as both observation and 
experiment show. Similarly, is it hazardous to compare 
the surface of the red blood corpuscle in human blood with 
that of human cells or even with the red blood cells of 
vertebrates other than mammals since the mammalian red 
blood corpuscle is not a true cell because it is devoid of a 
nucleus. Therefore, if the mammalian red blood corpuscle 
lacks a membrane this is no proof that true cells also lack 

The first proposition is thus clear: in defining a cell- 
membrane we must be careful to distinguish between the 
various coverings enclosing cells, excluding, for example, 
discussion of such formations as cellulose walls around plant 
cells. The second proposition is, likewise, obvious: if we 
speak of cell-membranes, we must speak of cells; the 
presence or absence of a membrane on a piece of a cell like 
the mammalian red blood corpuscle does not come within 
the discussion. The issue concerns cells and cells only. 
Indeed, much of the work on mammalian red blood cor- 
puscles should be used only most judiciously in making 
comparisons with true cells. 

Thus restricted, the discussion on the cell-membrane 
centres around two questions: Is there a membrane around 
animal cells ? And if present, is it a living part of the living 
system or an exogenous formation ? If exogenous, I 



presume that it is non-living. I think that the question of 
necessity or purpose is not involved. To say that one does 
not see how it is possible for a cell to remain intact without a 
membrane most certainly does not mean that it can not thus 
remain. Here I speak only of those cells that I know best, 
egg-cells, though 1 hold that the discussion can apply to 
some extent also to other animal cells which, like the eggs, 
are often held to be without membranes. 

In the description given above I pointed out that on eggs, 
as, for example, that of Platynereis, two membranes are 
present; the outer or vitelline, and the inner or plasma- 
membrane. Whenever egg-membranes are discussed, the 
two membranes should be clearly distinguished. Further- 
more, when the vitelline membrane is spoken of, it should be 
clearly stated whether the unfertilized or the fertilized egg 
is considered. Much of the disagreement concerning the 
nature and properties of egg-membranes is due to the 
confusing of the vitelline and the plasma membrane and to 
failure to appreciate the difference of the former before and 
after fertilization. 

Generally, the essential difference between a vitelline and 
a plasma-membrane lies in the fact that the latter is con- 
tinuous with the egg-cytoplasm whilst the former is dis- 
continuous with it, set apart from it and separated by a 
space, the so-called perivitelline space. Using this simple 
distinction we meet, however, with the difficulty that in 
many eggs that membrane which after fertilization is thus 
so clearly set off as a vitelline membrane, before fertilization 
appears as a plasma-membrane, continuous with the cyto- 
plasm. When after fertilization such a vitelline membrane 
separates from the egg, on the cytoplasmic surface of the 
egg appears a new plasma-membrane. 

In observing one living egg I may be able to say in a 
moment that a membrane is present, it being so sharply 
differentiated; observing another species of egg^ I may not 



be so sure but doubt vanishes if I know the history of the 
egg. Take the unfertilized egg of the sea-urchin concerning 
the presence of whose vitelline membrane there has been 
much dispute. Knowing the history of this tgg as it 
develops in the ovary I know that it arises by successive 
divisions, each egg everywhere bounded by a membrane 
except at its point of attachment to the ovaria-n wall. I 
know also that the egg is part of the parent whose cells arose 
by a long series of cell-divisions, of separations by cell- 
surfaces, from a fertilized egg. This history strongly 
indicates the presence of a membrane and the simple experi- 
ments made previously by others which I repeat and con- 
firm strengthen the indications. What is true of this egg is 
true of all other eggs that I have studied: they possess 
membranes built by the egg, living structures and not 
adventitious depositions from the outside. In other eggs, 
those of worms, molluscs, ascidians as well as of other 
echinoderms, the presence of a membrane before fertilization 
can not be doubted. The fact that membranes become 
separated at a distance from the eggs after fertilization does 
not mean that the eggs are now without plasma-membranes 
for they at once form them anew. Careful observation 
can establish this as true. 

It follows from these considerations that the membrane 
is neither a deposition nor a precipitation induced by the 
egg's surrounding medium. This is not to say that the 
egg-plasma never forms precipitation-membranes. On the 
contrary, the contents of an egg caused to burst by applica- 
tion of pressure may often show a congealed surface. But 
this death-change by no means constitutes evidence that 
the membrane on the living intact egg is a precipitation- 
membrane. A pared apple in time develops by dessication 
an outer tough coat but this is not the same structure as the 
removed skin. Nor does it follow on the definition of 
protoplasm Ct as a film-pervaded system/ 1 which is often set 



forth, that the external covering of a cell is similar to or 
identical with such intracellular partitions. 

The understanding of the cell-surface and of the capaci- 
ties residing in the ground-substance is forwarded by 
appreciation of that component of the protoplasmic system, 
which I now discuss in detail: The ectoplasm. 


The Ectoplasm 


sponges, first used the terms, exoplasm and endoplasm. 
He clearly distinguished two regions of the cytoplasm as 

In the hyaline contractile ground-substance of the proto- 
plasm a varying mass of small dark granules is constantly 
embedded; these usually surround the nucleus. On the 
living flagellar cell, as long as it is active in situ, is a thin 
granule-free cortical sheath. Thus one can more or less 
clearly distinguish between a structureless outer cortical 
substance and a granular inner medullary substance. The 
outer cortical substance (exoplasma) is fully hyaline, some- 
what firmer, less watery, more strongly refractive and 
contains no granules; the inner medullary substance (endo- 
plasm a) is granular, somewhat softer, more watery, less 
refractive (and contains the granules) and also now and 
then vacuolar. No matter how distinctly the regions some- 
times are set off from each other, they are never sharply 
separated but pass insensibly the one into the other without 
fixed limiting layer, much as is the case of hyaline cortex and 
granular medullary substance in the body of an infusorian. 

Later Haeckel says 2 that through its variously changing 
surface formation, the exoplasm gives to the entire flagellar 
cell its characteristic form. 

This clear description given by Haeckel may serve as the 
basis of our definition of the ectoplasm (exoplasm of 
Haeckel): the ectoplasm is the superficial region of the 
protoplasmic ground-substance set off from the remainder 

1 Haeckel, 1872. 

2 Haeckel, I.e. 



(both nuclear and cytoplasmic ground-substance) by differ- 
ences in physical properties, in structure and in behavior; as 
part of the cytoplasm, as differentiated ground-substance, 
it is definitely a living part of the cell. The inner region of 
the cytoplasmic ground-substance plus its cytoplasmic 
inclusions — -granules and the like — is defined as the endo- 

It is not to be supposed that ecto-endoplasmic differ- 
entiation in animal cells was unknown before HaeckePs 
time. Although not named as such, those regions were 
known to the older students of the cell. 1 Almost any 
original paper and text-book of theirs points out differences 
between the inner and the outer region of the extranuclear 
cell-contents. Thus, Henle in reports of his researches and 
Koelliker in his text-book on human histology gave clear 
descriptions of what subsequently were known as ectoplasm 
and endoplasm. Kidney, liver and intestinal cells were 
both described and figured to show these regions. The 
cells of the skin were favourite objects for such descriptions. 

If one observes a section of the kidney taken from man or 
other vertebrate one notes that certain cells are striated and 
possessed of radial projections. Thus, these cells reveal an 
ectoplasmic differentiation. 

The ectoplasmic area is less clearly marked off in the 
human liver cell. The manifold functions of the liver — the 
breaking down of red blood corpuscles, the formation of 
urea, the storage of sugar as glycogen, the mobilization of 
fat — are reflected in the many pictures of itself that the 
small polyhedral liver-cell can exhibit. It also appears 
differently in conditions of hunger and after rich nourish- 
ment. Nevertheless, in all its different appearances, the 
liver-cell shows a differentiation between inner and outer 
regions of the cytoplasm, as Koellicker observed. 

1 For example: Leydig, 1857; Kiihne, iS64. 



The cylindrical cells of the human intestine, measuring 
22-26 by 6 (x, show on the side presented to the intestinal 
lumen a brush border or bordure en brosse. Thus, in these 
cells as in those of the kidney and liver one notes an ecto- 
endoplasmic differentiation. 

In the skin are cells which interlock by means of fine 
threads, the so-called intercellular bridges. 1 Cells of other 
tissues are connected similarly, as for example those in the 
retina of the eye. Though many claim that these connec- 
tions are artefacts, the abundance of evidence from studies 
on various tissues from many different animals supports the 
view that these connections are normal cell-processes. As 
such they are evidence of ecto-endoplasmic differentiation. 2 

In view of the substantial body of convincing data 
showing the presence of intercellular connections, it 
becomes difficult to understand why many authors still deny 
their existence and assert that fixation creates them. But 
quite apart from the demonstration of these intercellular 
connections in well-fixed tissues exist clear cases, some of 
them among the earliest described, showing living cells in 
rich connections with each other. As will be shown later 
connections between cells of the developing egg have 
been long known and can be easily demonstrated. I think 
that almost no one to-day doubts that in the nerve-system 
the neurones are in contact with each other by processes 
which make the synaptic membranes. 

The body-structure of higher animals, especially of 
vertebrates including man, embraces certain groups of cells 
notable for the presence of extra-cellular substances asso- 
ciated with and produced by them. These groups of cells 
comprise the binding or connective tissues of the body. 
Their products are fibres of various kinds, cartilage and 

1 See Schuberg, 1903^ and later writers. 

2 Flemming, fSyg^ on living cells. 



bone. Studies on the development of fibres in connective 
tissue, on that of cartilage and of bone indicate that these 
structures which finally come to lie outside of the cell are 
products of the ectoplasm. Indeed, it has been postulated 
that connective tissue fibres even after deposition outside of 
the cell are living ectoplasm. Whether living or not, the 
intercellular substance of connective tissue and its deriva- 
tives (cartilage and bone) are to be regarded as extreme 
formations of ectoplasm, which have lost their organic 
connections with their cells of origin. 

Intercellular fibres cast off by cells are to be distinguished 
from such fibres which are true cells, having nuclei. These, 
muscle and some nerve cells, are characterised by their 
great length and hence by a preponderance of surface to 
mass. They thus possess relatively more living ectoplasm 
as part of the cells than any other cells so far discussed. 
Muscle-cells are notable for their high degree of contractility 
and students of physiology regard this as a surface-phe- 
nomenon. Fixed preparations of smooth or plain muscle- 
cells (individual fibres) show longitudinal threads or fibrils 
whilst striated muscle-fibres (both those of the heart and of 
the skeletal or voluntary muscle) show in addition striations 
running at right angles to the length of the fibres. Much 
difference of opinion is expressed concerning the significance 
of these fibrils because studies both on the developing and 
the definitive muscle-fibres have yielded conflicting results 
and led to various interpretations. In addition, many 
investigators doubt the actual presence of fibrils in 
muscle-fibres inasmuch as they have been unable to see 
such in living cells. Although it would seem hazardous to 
venture an opinion in view of this uncertainty with respect 
both to the longitudinal threads described in fixed smooth 
muscle and to the additional cross-striations reported in 
striated (cardiac and skeletal) muscle as seen in fixed 
preparations, nevertheless, I may offer suggestions concern- 



ing them. These suggestions are consistent with the well- 
established findings of studies made on both types of 
muscle-cells by the method of tissue-culture devised by 
Harrison and with the fact that in the muscle-cell the 
ectoplasm is preponderant. Also, they stand in accord with 
our knowledge of ectoplasmic structure in cells generally. 

The smooth muscle-fibre, an elongated slender spindle- 
shaped cell, reveals itself in fixed preparations as possessed 
of fine longitudinal threads, fibrils or myofibrils. Lewis and 
Lewis were unable with the use of either bright- or dark- 
field illumination to observe any fibrils in living smooth 
muscle-cells grown in tissue-culture. According to them, 
large flat cells seldom contract whereas the elongated and 
band-like ones sometimes exhibit rhythmic contraction. 
These latter show ectoplasmic processes. 

Very much elongated overlapping processes may simulate 
fibrils when parallel to the long axis of the cell. In many 
places the cells seem to spread out under considerable ten- 
sion, and it appears to be along the lines of tension that the 
contractile substance coagulates into fibrils of various sizes. 1 

From what is known of the contractile power of ectoplasmic 
processes of cells generally in tissue-culture, it is safe to 
conclude that the contractility of the smooth muscle-cell 
is likewise inherent. I suggest that the pictures of fibrils 
obtained in fixed preparations represent ectoplasmic pro- 
cesses somewhat altered by the action of the reagent 
employed to fix the cell. 

Striated muscle differs from smooth by showing alter- 
nate light and dark bands traversing its long axis. The 
exact structure of striated muscle remains in question. 
Since it begins its development as smooth muscle, it would 
seem most profitable to attempt an elucidation of its 
structure through study of its developmental stages. 

1 Lewis and Lezvis^ 1924. 



But in these studies is lack of agreement. According 
to one view the fibrils arise from cytoplasm which is 
itself of fibrillar structure; another view maintains that the 
fibrils arise from cytoplasmic inclusions. The living 
muscle-cell in tissue-culture often fails to show the structure 
seen in fixed preparations; according to many investigators 
cross striations do not appear until after the cell is fixed. 
I venture the following suggestion as to the cross-striations 
in the muscle-cell. 

The young striated muscle-cell, like that of smooth 
muscle, possesses ectopia smic prolongations. The pro- 
longations arising first fuse at their outer tips and thus 
form a membrane so that this region of the ectoplasm has a 
palisade-like structure. Repetition of this process through- 
out the length of the cells forms fibrils (the membranes) 
crossed by ectoplasmic prolongations. 

Beyond I shall show that the surface of the fertilized egg 
of the sea-urchin is made up of radial filaments covered by 
a thin membrane. Aly conception of the striated muscle- 
fibril has its origin in this fact. Since other cells than eggs 
show such projections, it is not too extreme to suggest that 
the embryonic muscle-cell also possesses such. The slender 
muscle-cells have the inherent capacity to build up succes- 
sively striated fibrils by reconstitution of their surface. 
The sea-urchin's egg builds one such surface layer which 
remains inseparably bound to the egg-cell; the muscle-cell 
builds many such surfaces. 

Also on other grounds my conception of the origin of the 
cross-striated muscle-fibril is not so far-fetched as it may 
seem. In the first place, the generally accepted doctrine of 
muscle-activity emphasizes the role of surface in the 
phenomenon of its contraction. The fact that in the 
contraction process, in one stage, oxygen is utilized, is 
evidence indicating a surface-reaction. Moreover, the 
shape of the muscle-fibre strongly supports the theory that 



its activity is limited to the surface. On more general 
grounds, it may be pointed out that it is held that muscle- 
contraction, ciliary action and amoeboid movement are 
similar and have a common basis. Ectoplasmic threads 
may be considered as fine amoeboid processes, delicate 
pseudopodia; a cilium may be regarded as a modified 
pseudopodium. On the basis of my suggestion the striae 
of a cross-striated muscle can be regarded as threads of 
ectoplasmic substance, and muscle as a strand compounded 
of pseudopodial processes. 

This suggestion of mine concerning the origin of the 
muscle-fibre from repeated building of sheets of ectoplasm 
is interesting in the light of a passage from Lewis and Lewis 
in their " Behavior of Cells in Tissues" 1 : 

There are three conditions resembling fibrillae, which are 
often observed, not only in heart-muscle cells, but in smooth 
muscle, endothelium, and mesothelium as well; namely, a 
linear arrangement of long mitochondria, an overlapping of 
long, slender processes, and tension striations. The latter 
deserve most consideration, because they resemble closely 
the appearance of fibrillae in fixed cells. Migrating cells 
become more or less flattened out on the solid supports 
under considerable tension. The direction of this tension 
appears to be in line with the cell processes, as though the 
latter produced a pull on the ectoplasmic layer. The visible 
striae of various widths and lengths thus produced in the 
living cell are not permanent but may disappear and new 
ones may appear in line with new processes. The exact 
significance of these striations is uncertain. They may 
extend across the nucleus, indenting it or almost cutting it 
in two. They seem to be a phenomenon produced by ten- 
sion and reversible when the tension is altered or relaxed. 
On fixation they may retain their identity and stain more 
deeply than the rest of cytoplasm, resembling myo-fibrillae. 
The latter occur however in fixed cells which do not show 
tension striae. 

Certain nerve-cells are also notable for their fibre-like 
nature. Among these are the longest cells known. One 

1 Lewis and Lezvis, I.e. 






* ' ', *w ,| 


Fig. 4. — (after Harrison), a y row of ectoderm cells showing amoeboid ectoplasm; 
by c and d are protoplasmic processes in developing nerve cells. 



cell and its process, for example, of the sciatic nerve in man 
which is made up of processes from nerve-cells in the spinal 
cord, may be easily a meter in length. What we speak of as 
a nerve is really a bundle of processes from nerve-cells. 
When, for instance, one makes experiments in the labora- 
tory on excised nerves — the sciatic nerve of the frog is a 
favorite object for such — one uses only the ectoplasmic 
portions of cells. Therefore, conduction by nerves means 
conduction by ectoplasm. That the nerve-fibre is ecto- 
plasm was fully demonstrated by Harrison's classic and 
epoch-making work on the origin of the nerve-fibre. 

Harrison's researches while establishing the mode of 
origin of the nerve-fibre also furnish a beautiful demonstra- 
tion of ecto-endoplasmic differentiation. Harrison, the 
originator of the now so widely used method of growing 
tissues outside of the body of an animal, had as his object 
the settlement of the much debated question of the mode of 
origin of the vertebrate nerve-fibre from the central nervous 
system. By growing young embryonic cells taken from the 
spinal cord of a frog embryo at a stage in which he knew 
that no nerve fibres had yet developed, he was able to prove 
that these cells extend pseudopodial processes which by 
farther growth become the nerve-fibre. Says Harrison 1 : 
(Fig. 4-) 

From the time when the tissue is implanted in the lymph 
it shows a tendency to spread out, and often broad laminae 
made up of a single layer of cells are found at the peri- 
phery of the mass, while individual cells may move off 
entirely by themselves. This is the case with both nervous 
and axial mesodermic tissue, as well as with pieces of ecto- 
derm, though the latter more often roll themselves into com- 
plete spheres. One notable peculiarity that has frequently 
been observed is the formation of large round or oval open- 
ings in the flattened tissue, which may be surrounded 
by very narrow bands or rings of tissue with cells sometimes 
in single file. This phenomenon may possibly be due 

1 Harrison, igio. 



to the mechanical action of the fibrin upon the implanted 
tissue, but the spreading out of the cells into thin sheets 
seems to result largely from the activities of the cells them- 
selves. These activities, which are common to several 
tissues, in fact to all except the very inert yolk-laden endo- 
derm and, perhaps, the notochord, may be referred to a 
form of protoplasmic movement having its seat in the 
hyaline ectoplasm found at the angles and sometimes at the 
borders of the cells. The movement cannot be observed 
clearly in the larger masses of cells on account of their 
opacity, but it may be seen very clearly in those cells which 
leave the main masses and wander off by themselves. 
These cells are irregular in shape, varying from unipolar 
form and having a varying amount of ectoplasm at their 
angles. The movement is amoeboid in character and 
results either in a change in shape of the cells or in their 
movement as a whole. Such cells are found usually in 
greatest numbers in preparations of the medullary cord, and 
from the cranial ganglia (branchial ectoderm), that gives 
rise by its movement to long fibres. Cells of the epidermis 
show their power of movement in somewhat different form. 
As has frequently been observed, the general tendency of 
isolated bits of epidermis is to round off into small vesicles, 
which, when left in water, may move about for days by 
means of their cilia. Within the lymph the same thing 
frequently takes place, although there is apparently 
greater resistance to the process of rolling up, and the 
cells may often remain together in the form of extensive 
sheets. Along the free border of these sheets of cells there 
often appears a fringe hyaline of protoplasm, which under- 
goes continuous amoeboid changes. In one case of this 
kind it was observed that the sheet of cells gradually 
spread out toward the side on which this fringe was placed. 
Since the work of Peters (1885— 1889) it has been generally 
admitted that wound healing in the epidermis is primarily 
due to the movement, in part amoeboid, of the epithelial 
cells, so that it seems quite possible that in this fringe of 
hyaline protoplasm above described, we have one part of 
the mechanism by which the movement of cells in wound- 
healing is brought about. The most inert of all the tissues 
is the endoderm, which will remain for days in the lymph, 
practically unchanged, gorged with yolk and devoid of hya- 
line ectoplasm. The notochord is also very inactive, 
although large pieces of this structure may show after a 

s 4 


time the early stages of normal differentiation, unaccom- 
panied, however, by growth, i.e., increase in length. 

. . . The movement of the embryonic cells in the lymph 
clot is very distinct, and is due beyond doubt to the activi- 
ties of the hyaline ectoplasm, which is accumulated espe- 
cially at the angles of the cells. It there forms extremely 
fine filamentous pseudopodia, through the activity of 
which the cells may change their shape or move from place 
to place. The exact character of the movement is not 
the same in all kinds of cells and it varies greatly in inten- 
sity. Axial mesoderm and medullary cord yield cells 
that frequently wander for considerable distances by them- 
selves; epidermis, when it does not roll up into bands or 
spheres, may form a hyaline fringe, and spread out con- 
siderably; pieces of the central nervous system and the 
primordia of the cranial ganglia give rise to the fibre-like 
structures described in the last section; the endoderm and 
notochord remain almost inert. 

In passing, I should like to refer to two points which 
baffle students of tissue-culture. I offer an interpretation 
of them on the basis of my knowledge of ectoplasmic 
behavior in general. 

The first concerns the observation so often noted that 
living cells in tissue-culture tend to assume a spindle- 
shape, especially when they migrate from the bit of tissue 
implanted in the culture-medium. There is no correlation 
between the original form of the cell and the spindle-form 
subsequently attained as we can conclude from the studies 
of many observers, who on various types of cells have con- 
firmed Harrison's original observations. Two suggestions 
have been offered: that the spindle-form is a reversion to a 
less differentiated (i.e., embryonic) cellular condition and 
that it is the effect of the cell's new, and abnormal, environ- 
ment. My suggestion is that the spindle-shape expresses 
gain in ectoplasm. I base this view on the fact that cells 
when isolated usually show relatively more active ectoplasm 
than when they are in contact and further on the observa- 
tion made by Harrison that spindle-shaped cells become 



extremely tenuous as they move along a delicate track, such 
as the thread of a spider's web, which behavior I interpret 
as a pseudopod extending in one direction. 

Concerning the cause of migration, two views are main- 
tained. According to one, the cells react positively to 
solids; according to the other, the cells move away from 
regions of too high acidity. Aly own interpretation is that 
cells in tissue-culture are no longer under the restraint 
imposed upon them when they stand in coordination with 
and subordination to their normal environment, i.e., other 
cells and tissue fluids. Thus their ectoplasmic activity is no 
longer ordered and controlled by the contact but is exagger- 
ated especially in their free and exposed surfaces — that is, 
in the regions where they are not in contact with the cells 
of the bit of tissue implanted on the medium. Hence, they 
migrate away from the mass. 

Suspended in the blood-plasma of vertebrates are red 
blood corpuscles. In mammals these are devoid of nuclei 
and therefore not true cells. All other vertebrates possess 
nucleated red blood cells. The structure of these latter has 
been worked out by Aleves, who found that they have a 
well-marked ectoplasm. 

The blood of vertebrates contains in addition to the red 
blood corpuscles the leucocytes or white blood cells. In 
human blood these cells are sub-divided into four kinds, 
cell-size, staining reaction of cytoplasmic granules, and 
nuclear form being the characters on which the sub-divisions 
are made. In a healthy adult individual there is a fairly 
constant number of each type of white blood-cell. This 
number is used as an index for pathological states — many 
diseases have each a characteristic " blood-picture" which 
is an important diagnostic aid. On these highly interesting 
cells ectoplasm can be easily demonstrated. It has largely 
been assumed that for phagocytic activity and movement of 
white blood cells the same theories hold that have been 



postulated for these activities in free-living Amoebae. 
But even were there only one accepted theory of the 
cause of amoeboid movement in free-living Amoebae, there 
would still remain the necessity to prove that such a theory 
could apply to white blood cells in the blood-stream of 
warm-blooded animals. It would be more advantageous 
further to study the white blood-cells themselves. Since 
amoeboid movement and capacity for engulfing solid par- 
ticles reside in the ectoplasm, 1 the study of primary im- 
portance for the elucidation of these phenomena exhibited 
by human white blood-cells relates to the ectoplasm of the 
various types of these cells and to each of them in their 
different physiological states in health and in disease. 

This cursory review dealing mostly with vertebrate 
tissue-cells by no means pretends at exhaustion. Tho- 
roughly to treat the subject of ecto-endoplasmic differentia- 
tion as found in cells of multi-cellular animals would 
demand a volume in itself. But the review shows that 
from sponges to man tissue-cells exhibit ecto-endoplasmic 
differentiation; that the ectoplasm gives rise to processes 
which interlock with other cells; that the ectoplasm may be 
cast off to form inter-cellular substance; that it enters 
largely into the formation of contractile cells (muscles) ; that 
it is the conductive material par excellence of nerve-cells and 
that cells, as amoeboid blood cells, in their locomotor 
capacity indicate ectoplasmic action. 

Ecto-endoplasmic differentiation is by no means confined 
to the tissue-cells of multicellular animals. Very early in 
the history of the study of Protozoa, it was recognized that 
in these unicellular organisms the superficial cytoplasm is 
marked off structurally from the interior. Indeed, nowhere 
else in the animal kingdom is the ectoplasm so sharply 
defined by so rich and various sculpture. 

1 Metschnikoff on amoeboid cells of sponges, 1879. 

S7 ' 


To Haeckel we owe the application of the term, exoplasm, 
to the superficial cytoplasm of the Protozoa. Writing in 
1873 on the morphology of the Infusoria, he emphasized the 
differentiation of the protoplasmic body into a bright, 
firmer cortical substance and a granular, softer medullary 
substance, a differentiation found also in Amoeba as in 
many parenchyma cells of higher animals. This outer 
layer is further distinguished from the medullary portion of 
the cytoplasm through a lower water content and an 
independent contractility. Later authors, as Biitschli, 
Doflein and others, have merely reiterated HaeckePs 
original description, confirming it through their own 

The protozoan ectoplasm may be highly differentiated: 
for support and protection; for motility; for food-capture; 
and for oral and anal modifications. In it are located the 
contractile vacuoles. On the basis of their means of 
locomotion Protozoa are classified into four groups: those 
like the Amoeba, that move by means of pseudopodia; 
those that move by means of flagella; those like Para- 
moecium that move by means of cilia, and, in a negative 
way, those like the Sporozoa, to which tfre organism that 
causes malaria belongs, which are characterized at least 
during one period of their life-history by lack of locomotor- 
apparatus. In other words, the classification of the groups 
is built upon ectoplasmic differentiation. 1 

1 Though this book concerns itself with the animal cell, I may note 
in passing that in many plant cells, especially in the higher, multi- 
cellular, organisms, the living cytoplasm is entirely superficial in 
location, the interior of the cell being a vacuole of non-Inn ng materials. 
Also, I may call attention to the Bacteria which possess a strongly 
marked ectoplasm, investigated especially by Gutstein (1926 and 
earlier). The ectoplasm of Bacteria has been much discussed in 
connection with their structure in relation to the valuable diagnostic 
aid for identifying these organisms furnished by the Gram stain. 
{See Schumacher 192S and earlier.) 



While the ectoplasm of the Protozoa has been well known 
for years, that of eggs has not been generally recognized, 
albeit eggs from every phylum in the animal kingdom either 
have been described as showing ectoplasm or can be shown 
to possess such. Since here we are concerned primarily 
with eggs, it is necessary to review in detail the evidence 
with respect to their ectoplasmic differentiation. 

The terms, exoplasm and endoplasm were first used by 
Haeckel to describe the outer and inner cytoplasmic regions 
of the sponge egg. In his monograph on the sponges 
referred to at the beginning of this chapter, he speaks of the 
hyaline exoderm (exoplasma) and a granular endoplasm 
sharply set off from it. Metschnikoff 1 in describing 
eggs of a sponge emphasized its changes as due to amoe- 
boid processes of the surface-cytoplasm. Gatenby 2 has 
described the ectoplasm of the sponge Qgg as follows: 
(Fig. 5). 

Even in the youngest oocytes one may notice that at an 
early stage a clear ectoplasmic zone becomes differentiated 
from an inner or endoplasmic zone. The ectoplasmic zone 
contains few or no vacuoles, is smooth, and is often drawn 
out in the form of blunt pseudopodia-or filamentous dendri- 
form threads. The inner or endoplasmic zone is vacuolated 
completely and has a fine, frothy appearance; it is in this 
region that the cytoplasmic inclusions lie, granules in the 
ectoplasm of the oocyte being rare or never found. Occa- 
sionally, in preparations fixed in mixtures containing alcohol 
or acetic acid, the vacuoles collapse, and the egg comes to 
have a curious radiation of fibres around the nucleus (see 
Joergensen's figures 8a). Eggs treated with silver nitrate 
solution show the endoplasm browner than the ectoplasm. 
... In favourable cases not only young oocytes, but 
amoebocytes, may be seen to possess ectoplasmic and 
endoplasmic zones. As in the case of the older oocyte, the 
cytoplasmic inclusions lie in the endoplasm, while the 
pseudopodia consist mainly of ectoplasmic material. 

1 Metschnikoff, I.e. 
-Gatenbv, 19T9. 



During development of the egg, all the blastomeres in 
most cases come to have an equal portion of the ectoplasm. 

The ectoplasm can be traced through cleavage up to the 
formation of a blastula, but it soon either becomes absorbed 

Fig. 5. — Egg of a sponge, Grantia (after Gatenby) showing amoeboid ectoplasm. 

or is invaded by endoplasmic substance. ... In a rare 
number of cases, it was found that in young blastulae the 
cells of one side had less ectoplasm than those elsewhere 
but in no example could I show that this inequality had 
any relationship to the formation of the flagellated and 
non-flagellated parts of the amphiblastula. 

# . v . : -■■-;-..<! tM 


i ■.;-■■• 

V^ : 

y V: 


a b c 

Fig. 6. — Eggs of medusae (after Metschnikoff). a, Liriope mucronata; b, and 

c, Rathkea. 

In the cytoplasm of eggs of all classes of Coelenterates, 
including the Ctenophores, that is, the jelly fishes and their 
allies, ecto-endoplasmic differentiation has been frequently 



noted. (Fig. 6 and Fig. 7.) Kowalewsky 1 without naming 
the two regions gave a very clear description of them as 
found in several ctenophore ("comb-jelly") eggs. Sub- 
sequently, authors have confirmed Kowalewsky, 2 often 
only repeating the words of this great observer. Says 

The yolk mass of the eggs of Idyia (and all Ctenophorae) 
consists (after they are laid) of two layers, an inner yolk 
mass more or less fatty, made up of large, irregular spheres. 
This inner yolk mass is surrounded by a second thin, 
outer layer, finely granular. This outer layer and its 
enclosed central mass perform very different parts in 
the development of the embryo, and it is of the utmost 
importance to keep the changes these two layers undergo, 
clearly distinct, while following the development of the 
young Ctenophore. The outer layer as has been shown by 
Kowalewsky, is eminently the embryonic layer, while the 
inner mass acts as a mere nutritive yolk mass. 

Fol 3 also described ecto- 
endoplasmic differentiation in 
Coelenterate eggs, using the 
term, ectoplasm, instead of 
Haeckel's exoplasma. Perhaps 
the most striking ectoplasmic 
structure described in eggs is 
that found in the Beroe-egg 
noted first by Chun and later Fig. 7. — Egg of a ctenophore, 

by YatSU. 4 The ectoplasm in Escholtzia (after Kowalewsky). 

this egg is of a bright green colour, a fact that one 

1 Kowaleivsky, 1866; iSyj. See also Kozualewsky and Marion, 


2 Agassiz, 1874. 

3 Fol, 1873. 

4 Chun, 1880; Yatsu, S907. For other observers on the ectoplasm 
of Coelenterate eggs see: Metschnikoff, 1886; Ziegler, 1898; Appelof, 
1900; Hargitt, 1904; Spek, 1926; Conklin, Cam. PubL, IOJ; 
Ciamician, 18/p; Korotneff, /88p; Harm, 1903; Maas, 1908; 
Torrey, 190J; Komai, 1922. 



can easily confirm under low power of an ordinary 
microscope. Indeed, I have with the naked eye seen the 
green cytoplasm on this rather large egg; also have I 
found that under pressure the endoplasm, which Kowalew- 
sky did not consider living substance, will stream out as 
the large egg ruptures leaving the green surface-cytoplasm 

The difference between the peripheral and the inner cyto- 
plasm of eggs of flat-worms has been often observed/ 
though some authors fail to do so, and still others have 
expressed the opinion that the structural differentiation 
observed might be due to fixation. 2 Many flat-worm eggs 
present serious difficulties for study in the living state 
because they are laid in capsules each containing several 
eggs; removal of the eggs from the capsule may mean their 
injury. In addition, because of opacity, they do not always 
lend themselves to observation of their finer structure. 
Nevertheless, one may assert that eggs of turbellarians and 
trematodes and even of those of cestodes (tape-worms) 
exhibit a resolution of the cytoplasm into an endoplasmic 
and an ectoplasmic region, as Metschnikoff, Selenka, Lang, 
Pereyaslowzewa and Janicki, among others, have shown. 

The thread-worms, nematodes, include Ascaris, a para- 
sitic worm which has been the object of many researches 
and which will come up for reference frequently in this 
book. The unfertilized egg of Ascaris shows, accord- 
ing to Van Beneden and Meves, ectoplasmic differen- 
tiation; irregular in shape, after fertilization it becomes 
spherical having in the meantime secreted a substance 
which forms an enclosing shell. The eggs of free-living 
nematodes, closely allied to Ascaris, show a similar change; 

1 Metschnikoff, /SSj; Selenka, iSSj; Lang, /SSj; Pereyaslowzewa, 
1S92; Warren, 1903; Janicki, 190J. 

2 Halkin, 1902. 




on fertilization, bubbles in the cytoplasm break through the 
surface which undergoes amoeboid changes, then the eggs 
attain a regular contour. 1 Cerebratulus, a long ribbon-like 
worm found in the sea, lays eggs in which the cytoplasmic 
granules in the endoplasm differ from those at the periph- 
ery. 2 This egg also displays remarkable amoeboid changes 3 
which always speak for the 
presence of an ectopias mic 

Eggs of the wheel animal- 
cules or Rotifera according 
to several investigators show 
a distinct ectoplasm. 4 

In Yatsu's interesting ac- 
count of the development of 
the egg of the mollusk-like 

£>*r. :->*VV 

animal, Lingllla* is a de- Fig. 8.— Egg of Lingula anatina (after 

tailed description together latsu). 

with pictures showing the surface-located cytoplasm set off 
from the endoplasm. (Fig. 8.) According to Prouho, 6 the 
egg of Membranipora possesses delicate projections which 
he figures. 

In the unfertilized eggs of the echinoderms (starfishes, 
serpent starfishes, sea-urchins, sea-cucumbers, sea-lilies) 
the ectoplasm is well defined. Selenka 7 has described the 
changes which a sea-urchin egg undergoes before it 
reaches the stage when it is ready for fertilization. These 

1 Biitschli, iSjj; also v. Erlanger, iSgj and later authors. 

2 Coe, iSgg. 

3 Andrews, E. A., /Sgj; Wilson, C. B., iSgg and others. 

4 Zelinka, iSg2; Siorch, 1924 and others. 

5 Yaisii, 1902. 

6 Prouho, 1S92. 

7 Selenka, 1S7S. 



Fig. 9. — For descriptive legend see page 95. 



changes largely concern the ectoplasm, as the appended 
figures (9) show. Though I have sought to obtain these 
stages in other species of sea-urchins, I have so far failed. 
In mature echinoderm eggs the ectoplasm, though difficult 
to observe, can be made out in the living egg as a homo- 
geneous extremely narrow rim beneath the vitelline mem- 
brane. In fixed preparations it is only very faintly marked 
off from the endoplasm. After fertilization the ectoplasm 
stands out clearly because of the now present hyaline 

Fig. 9. — Development of the ectoplasm in the egg of Toxopneustes variegatus 

(after Selenka). 



plasma-layer which though it has often been described 1 
(Fig. 10) has never, as we shall see in the chapter on cell- 
division, been properly interpreted by those who have 
observed it, and generally been misunderstood by those 
who have on the basis of experiments on it made theories 
concerning its role. For eggs of the brittle starfishes and 
of the sea-cucumbers, Selenka has given good descriptions, 

pointing out that the layer is 
most pronounced in eggs of the 
brittle starfishes, less in those 
of sea-urchins and least in sea- 
cucumbers'. I find that in 
these as in the eggs of the 
/ starfish the ectoplasm can 

Fig. io. Fig. ii. 

Fig. io. — Egg of Echinocyamus pusillus (after Theel) to show structure of 
ectoplasm after full separation of vitelline membrane following penetration of 
a single spermatozoon. 

Fig. i I. — The ectoplasm of the egg of Rhynchelmis limosella (after Vejdovsky 
and Mrazek). 

always be distinguished in sectioned eggs fixed with certain 
reagents for then minute granules appear; also the delicately 
radial structure is evident. 

I turn now to the eggs of the segmented worms. Very 
strikingly does the ectoplasm differ from the endoplasm in 
the egg of Rhynchelmis 2 as pictured here (Fig. n). But no 
less clearly or remarkably do the inner and outer regions in 
the egg of Phascolosoma? reveal themselves. The ecto- 

1 Hertwig, 1S76; Selenka, 1S7S; Fol, 1S79; Ludwig, 1SS0; 
Selenka, iSSj; Berthold, 18S6; Theel, 1S92; Hammar, 1S96; 
Andreivs, 1S97; Ziegler, 1904; Meves, IQ14. 

2 Vejdovsky, 1S92; Vejdozvsky and Mrazek, 1903. 
*Gerould, 1907; Bergmann, 19OJ, on annelids. 



plasmic structure of the Rhyncheimis egg is similar to that 
recorded for the Nereis-egg by several workers beginning 
with Goette 1 and this in turn recalls SpengePs older descrip- 
tion for the egg of Bonellia. 2 
Also the Chaetopterus-egg has an 
interesting ectoplasm, 3 so too that 
of Thalassemia^ (Fig. 12). 

Among the great group of soft- 
bodied animals, the mollusks, 
including the clams, snails and 
ink or cuttle fishes, the following 
produce eggs in which the ecto- 

tig. 12. — Ectoplasm on the 

plasm has been observed to be n v i ng egg of Thallassema mellita 

marked off from the endoplasm: (after Lefevre). 

Mactra r ° and Mytilus^ (clams), various species of Chiton, 1 

Patella* (a snail) (Fig. 13), Dentalium^ (a scaphopod) and 
the cuttle-fishes (cephalopods). 10 In the eggs of Mactra 

1 Goette, 1SS2; v. Wistinghausen, iSqi; Wilson, 1S92; Hempel- 
niann, 1911; Lillie, P. R., 1912. 

2 Spengel, 1S79. 

3 Li Hie, P. R., 1906. 

4 Torrey, 1900; Lefevre, 190J. 

5 Kostanecki, 1904. 

6 Meves, 1915. 

7 Koivalewski, 18/S. 

8 Patten, 1SS5; Jorgensen, 1913. 

9 Wilson, 1904. 

10 Bergmann, I.e. 



the outer sheath of cytoplasm contains large granules, 
whilst in that of My til us according to Aleves' figures the 
ectoplasm in the fixed egg is a clear margin bounded by a 
homogeneous and apparently firmer sheath of cytoplasm 

beneath the very thin vitelline mem- 
brane. In figures of the eggs of Chiton 
also one distinguishes ectoplasm from 
endoplasm. In addition, my observa- 
tions convince me that in the eggs of the 
razor clam, Ensis, and of the small clam, 
Cumingia, the outer region of the cyto- 
plasm differs from the inner. The large 
ellipsoid eggs of cephalopods — ink-fishes 
and allies— show a disc of clear cytoplasm 
containing the nucleus at the upper more 
pointed end which is continuous with a 
very thin layer enclosing the inert yolk. 
Disc and layer constitute the active sub- 
stance of the egg; in them development 
ensues; it is as though the cytoplasm in 
these eggs were all ectoplasm. 

Among eggs of arthropods are those of 
insects, 1 whose ectoplasm is even more 
strongly marked than that in eggs of 
Fig. 14.— Egg of Hy- coelenterates. Beyond we shall note 

drophilus pisceus (after . r 1 1 

He j der A that the ectoplasm 01 these eggs has most 

interesting behavior. Appended is a 
picture from Heider showing the ecto-endoplasmic differ- 
entiation in the egg of a beetle (Fig. 14). Eggs of other 
arthropods clearly show ectoplasm. 2 

Among the lowest forms of Chordates, the Ascidians, 
we find that the beautifully transparent egg of Phal- 

1 Heider, 1889. 

2 Groom, 1S94. 



lusia 1 reveals ecto-endoplasmic differentiation. The egg of 
Amphioxus 2 likewise has an outer sheath of cytoplasm 
marked off from the inner (Fig, 15). The ectoplasm 
of the latter egg as pictured by Sobotta recalls that already 
above described for the egg of Nereis or of the sea-urchin. 
That all other authors do not note cytoplasmic differ- 
entiation in the egg of Amphioxus one may attribute 

either to oversight or to the quality of fixation. The 
separation of the vitelline membrane exemplified by this 
egg constitutes additional evidence for similarity of its 
ectoplasmic structure to that of the other eggs named. 
However, this membrane separation has seldom been 
observed because of the difficulty of securing the early 
fertilization-stages for observation. 

1 Aleves, igi3. 

2 Sobotta, 1S9J; Cerfoniaine, 1906. 



Ascidians and Amphioxus belong to the chordates with- 
out back-bone or vertebral column. Fishes, amphibians, 
reptiles, birds and mammals are chordates with a vertebral 
column. The lamprey is an example of the low vertebrate, 
having no true jaws to the mouth as other vertebrates have. 

Fig. i 6. — Egg of Petromyzon fluviatilis (after Her fort). 

Its egg has been described by several investigators, among 
these Calberla, whose beautiful work on fertilization of the 
lamprey egg is classic. According to him this egg shows 
regional cytoplasmic differentiation. 1 See also Herfort, 
(Fig. 16.) ' 

For eggs of selachians (sharks and their allies), His has 
written as follows: 

1 Calberla, iSjj; Bohm, fSSS; Herfort, igoo. 

i no 


It thus seems practical to speak instead of kinds of 
plasma, of zones of the cell-body and we can distinguish: 
A central condensed zone. ... A zone of network. ... A 
hyaline cortical zone. . . . l 

In the egg of teleosts (bony fishes) the protoplasm, at 
first a thin sheath enclosing the yolk, moves at fertilization 
to one pole to form a disc leaving only a delicate layer to 
surround the remainder of the yolk mass. 2 Here the rela- 
tions are as in the eggs of cephalopods. 

Living eggs of amphibians (frogs, toads, salamanders) 
have been long recognized as showing a differentiated 
surface due to the presence of pigment extending over 
approximately one-half the egg. In sections amphi- 
bian eggs show further differentiation of the superficial 
cytoplasm. 3 

In the eggs of both reptiles and birds the protoplasm is 
sharply set off from the large mass of inert yolk. The 
description given by His for the selachian egg and quoted 
above applies to the eggs of reptiles and birds. 

Recently, Lewis and Hartmann have described the 
organization of the living egg of the monkey. 4 Other eggs 
of mammals, as that of the mouse, bat, rabbit, etc., also 
show some difference between the peripheral and the 
central cytoplasm. 

In addition to these accounts which describe eggs of 
every group of animals as having ectoplasm, we should note 
those studies which show that ectoplasmic structure is 
revealed by experimental treatment. Hammar's descrip- 
tion of the ectoplasm on the sea-urchin's egg as composed of 
radial striations was based on observations on eggs in 

1 His, 1897. 

2 Ltsi, 1S87. This process was very beautifully described as 
early as 1S54 by the English physician, Ransom. 

3 King, 1901. Earlier: Goette, 1875. 

4 Lewis and Hartmann, /pjj. 



sea-water made more concentrated by evaporation. In 
calcium-free sea-water, the ectoplasm of sea-urchins' eggs 
fairly bristles with delicate filaments. In other eggs also 
the presence of these filaments can be demonstrated by 
experimental treatment — especially by placing the eggs in 
hypertonic sea-water. It is to be emphasized that the 
experimental means does not cause the formation of these 
filaments but only makes them more easily visible. Thus, 
in eggs of Nereis and of Platynereis the ectoplasmic fila- 
ments, seen in the unfertilized eggs as ectoplasmic striations 
and for a short time after fertilization as strands, can always 
be more strongly revealed in later stages, when they are 
seen with greater difficulty, by submersing the eggs in 

sea-water made hypertonic by the 
addition of NaCl or KC1. The same 
observation has been made on eggs of 
other worms, for example, on those of 
Sabellaria (Fig. 17). The often ex- 
pressed opinion that the ectoplasmic 
prolongations thus made visible are 

Fig. 17. — Egg of Sabel- r , • 1 i 1 r 

laria aheolata to show artefacts leaves unconsidered the fact 
effect of hypertonic sea- that the eggs show normally an ecto- 
water on the ectoplasm plasmic structure, as has been abun- 

(after Faure-Fremiet). 

dantly proved for eggs from those of 
sponges to those of vertebrates. The descriptions in the 
literature admit of no doubt on this point. 

When we place alongside this fact the observations 
detailed by students of tissues in culture on the strongly 
expressed ectoplasm, the generally accepted fact of ecto- 
plasmic structures in Protozoa together with the evidence 
of intercellular connections between cells in tissues and the 
knowledge of the mode of union of nerve-cells by processes, 
we have very good reason for asserting that the filaments 
and prolongations of the ectoplasm are its characteristic, 
and perhaps its most important, structure. They have 



often been compared to protozoan pseudopodia, especially 
to those that are extremely tenuous and fine. Years ago 
in a work that never has received adequate attention, 
Mrs. Andrews 1 emphasized the capacity of these threads for 
ceaseless changes which she spoke of as a spinning activity 
of the protoplasm. 

The tremendously impressive fact of the existence of ecto- 
plasmic structure finds here for the first time its proper 
appreciation. A structure which can rightfully be denom- 
inated a sine qua non of living matter can be presumed 
to have significance for the grand problem of biology, 
the revelation of vital phenomena. In the following pages 
I aim to establish the thesis that in ectoplasmic behavior 
we witness the expression of activities that set apart the 
living thing from the non-living, that mark how life main- 
tains itself ever in harmonious tempo with the ceaseless 
changes in its surroundings. 

1 Andrews^ Lc. 


General Properties of the Ectopias/// 


what is life? we can say what are the manifestations of the 
state of being alive. Respiration, conduction and contrac- 
tion are fundamental properties of living matter, exhibited 
by all living cells. Life never appears without them. 
Whilst inanimate things as well as organisms after death 
may display properties like these, only the living thing main- 
tains them in self-regulation through continuous adjust- 
ment. Now the question arises: to what extent are these 
biological indices, these manifestations of life, properties of 
the ectoplasm? Study of the egg's behavior in the first 
moments after sperm-contact will throw light on this 

The attachment of a single spermatozoon to an egg and 
the effect produced thereby can best be observed micro- 
scopically by adding a drop of sea-water containing very 
few spermatozoa to sea-water that contains eggs. To eggs 
of Arbacia spermatozoa attach themselves within two 
seconds after they are added to the sea-water containing 
the eggs. Under the impact of a spermatozoon the egg- 
surface first gives way and then rebounds; 1 the egg-mem- 
brane moves in and out beneath the actively moving 
spermatozoon for a second or two. Then suddenly the 
spermatozoon becomes motionless with its tip buried in a 
slight indentation of the egg-surface, at which point the 
ectoplasm develops a cloudy appearance. This turbidity 

1 Cf. Derbes. L c. 



spreads from here so that at twenty seconds after insemina- 
tion — the mixing of eggs and spermatozoa — the whole 
ectoplasm is cloudy. Now like a flash, beginning at the 
point of sperm-attachment, a wave sweeps over the surface 
of the egg, clearing up the ectoplasm as it passes; careful 
observation reveals that the ectoplasm now shows a brush- 
work of threads beneath the membrane. Twenty-five 
seconds after insemination, a cone of ectoplasm protrudes 
from the egg and encloses the sperm-head. This is sud- 
denly pulled into the cone; the ectoplasmic threads break at 
the site of sperm-attachment and release the membrane. 
Progressively from this point the membrane separates in a 
wave from the surface of the egg, leaving in its wake collaps- 
ing ectoplasmic strands. Thirty seconds after insemination 
the membrane is separated from the egg by a narrow peri- 
vitelline space. During the ensuing twenty-five seconds 
this space increases in width; the ectoplasmic strands 
become more sharply defined giving the ectoplasm the 
appearance of a striated layer. The vitelline membrane 
becomes equidistant from the egg at all points and the 
perivitelline space is at its greatest width one hundred 
twenty seconds after insemination. 

This progressive lifting of the membrane, first noted by 
Derbes, 1 I have studied in several species of eggs in great 
detail. 2 On the basis of experiments with physico-chemical 
means which induce membrane-separation, certain investi- 
gators have simply assumed the de novo origin of the mem- 
brane which they called the "fertilization-membrane." 
However, careful observation shows, as described above, that 
the membrane is already present on the egg and separates 
at fertilization by a progressive wave beginning at the point 
of sperm-entry. It thus is not a " fertilization-membrane," 

1 Derbes, 1S4.J. See also Fol, 1S79. 

2 Just, 1919a, 192 1, I922e, 1928c and f, 1929a and b. 



but properly the vitelline membrane. I have repeatedly 
followed the process in the egg of Arbacia, though it is not 
the most favorable form for this study. In the eggs of the 
genus Strongylocentrotiis, of Echinus and of Echinarachnius, 
for example, the separation of the membrane is more easily 

If eggs of Echinarachnius be inseminated with thin sperm- 
suspension, they throw off their membranes that are 

d e 

Fig. i 8. — Successive stages of membrane-separation in the living tgg of Echi- 
narachnius parma, site of sperm-entry. 

fully lifted and equidistant from the egg-surface in about 
two minutes. Membrane-separation follows sperm-pene- 
tration. After the sperm-head has entered the ectoplasm, 
a blister appears on the egg-surface at the point of sperm- 
entry. This blister contains drops that move toward the 
membrane and go into solution. The process is gradually 
continued throughout the whole ectoplasm, and thus the 
membrane is lifted in a wave that sweeps over the surface 
(Fig. 1 8). By break-down of material in the ectoplasm, 
beginning at the site of sperm-entry, the membrane is 
separated from the surface. After this membrane is fully 



off the egg, the ectoplasm again builds a new surface-struc- 
ture, the hyaline plasma-layer. 

Before the actual separation of the vitelline membrane, 
an ectoplasmic change beginning at the point of sperm- 
entry sweeps over the egg which immunizes it to other 
spermatozoa, the pole opposite the site of sperm-entry 
being the last point affected. The site of sperm-entry 
becomes a "point of injury" and is "negative" to the entry 
of spermatozoa arriving at this point, all other points around 
the egg for a brief moment being "positive." Especially 
in slowly reacting eggs it can be noted that this "wave of 
negativity ?: moves over the egg, at a rate which varies 
with that at which the sperm-head disappears within the 
ectoplasm. When only the tip of the sperm-head has 
entered the ectoplasm, the immediate vicinity of the site 
of penetration can not engulf another spermatozoon. As 
more of the sperm-head disappears within the ectoplasm, 
the "negativity" to sperm-entry progresses still farther 
around the egg; when the head has disappeared, the egg 
can engulf sperm only at one point — the pole opposite 
that at which the spermatozoon entered the egg. The 
"wave of negativity" thus precedes the actual beginning of 
membrane-separation. Before the membrane begins to 
separate at the site of sperm-entry, other spermatozoa can 
no longer enter at any point on the egg. From the point of 
sperm-entry a definite gradient of membrane-separation is 
established. This gradient, therefore, follows that of loss 
of susceptibility to sperm-penetration. 

When a spermatozoon becomes attached not only the 
penetration of other spermatozoa but also their fixation to 
the egg depends upon the degree of penetration of the effec- 
tive spermatozoon, that is, on the rate at which the "wave 
of negativity" is propagated around the egg. Thus, at the 
beginning of penetration, other spermatozoa can not become 
attached to the egg in the immediate neighborhood of the 



point of sperm-entry. Those farther removed may become 
attached, lashing back and forth very vigorously, until 
with farther penetration of the one successful spermatozoon 
the "wave of negativity 51 reaches them and their movement 
comes to a stand-still. 1 

The ectoplasmic changes in the egg of Nereis that take 
place after sperm-attachment are also worthy of note. 
Before insemination the ectoplasm of this egg is a broad 
chambered structure. When eggs and spermatozoa are 
mixed, -sperm-attachment rapidly ensues; then follows the 
escape of material from the ectoplasm, which rapidly sets 
in the sea-water as a transparent jelly. The ectoplasmic 
chambers break down, leaving only strands that cross the 
space between the inner part of the egg and the membrane. 
Now comes a period of amoeboid changes in the egg: 2 it 
shrinks, becomes markedly irregular in outline, its contents 
become darker and the perivitelline space much reduced. 
When this period is over, the egg assumes a clearer and more 
rounded appearance; the perivitelline space widens again. 
The delicate plasma-membrane beneath the vitelline mem- 
brane is now easily visible. 

In this egg, thus, the ectoplasmic changes differ from 
those observed in the eggs of Arbacia and of Echinarachnvus. 
In all three however the essential response to insemination 
is the same, namely, an alteration in the ectoplasm. These 
changes have significance for fertilization and experimental 
parthenogenesis as we shall see. At present, another aspect 
of them elicits our interest. 

By experiments with dilute sea-water, I was able to cor- 
relate the structural changes at the surface of the eggs 
named above with an alteration in the physical state of the 
e gg- I found, for example, that the egg of E chin arachnitis 

1 Just, 1919. 

2 Cf. Torrey y 190J on amoeboid changes in eggs of Corymorpha. 



is many times more susceptible to dilute sea-water during 
the period of membrane separation than the fertilized 
egg before or after this separation, or than the unfertilized 
e gg- The egg is never again so susceptible, although 
during the cleavage-cycles it exhibits rise and fall in 
susceptibility which may be correlated with the rhythm 
of nuclear division. We may consider in detail this sus- 
ceptibility to hypotonic sea-water first as seen in the egg 
of E china r achnvu s . 

If to a drop of sea-water containing unfertilized eggs of 
E chin arachnitis mounted under low power of the micro- 
scope, tap or distilled water be added, one can observe that 
the eggs take up water, swell, and finally break down in 
about two minutes. For example: the time of disintegra- 
tion in tap water for lots of eggs from ten females was as 
follows : 

Number of females I I 21 3 

Time in seconds to disintegration of the [ 

eggs while in tap water I270 243 60 


150 148 






The rate of disintegration in eggs from these same females 
exposed to the action of tap water, five to ten seconds after 
insemination, was about the same. Eggs from the same 
females exposed to the action of tap water two minutes 
after fertilization, i.e., after the membranes are completely 
off the eggs, withstood the exposure even better than the 
unfertilized eggs. But with the beginning of membrane 
separation, twenty to thirty seconds after insemination, 
the picture is quite otherwise, for then the eggs are highly 
susceptible. The following table gives the results of such 
an experiment made on eggs of the same females as were 
used in the above described experiment. 

Once it was well established by experiment with tap 
water that the period of high susceptibility falls in exactly 
with the period of membrane separation, attention was 



Number of females 

Time in seconds to disinte- 
gration of eggs exposed to 
tap water during mem- 



19 20 




directed to the susceptibility of the egg to less dilute sea- 
water at different stages of the process of membrane separa- 
tion. In the experiment with the tap water it appeared 
that when the egg cytolyzed, the break always came in that 
part of the ectoplasm from which the membrane was lifting 
at the time of exposure. However, the cytolysis induced 
by tap water was far too rapid to allow exact observations 


Fig. 19. — Diagrams showing disruption of egg-contents at site of membrane- 
separation in egg of Echinarachnius par ma, a is a fertilized egg in which mem- 
brane-separation has not begun. 

of this phenomenon. The experiments with less dilute 
sea-water proved that the observation made during expos- 
ure to tap water was correct: when eggs are exposed to 
dilute sea-water during the period of membrane-separation 
they cytolyze by an outflow of cytoplasm at the points from 
which the membrane is lifting at the moment of exposure 
(Fig. 19). Thus the susceptibility travels at the same rate 
as the wave of membrane-separation. As stated above, 
membrane-separation in the normal process results from 
the solution of substances in the ectoplasm. Under the 
microscope one can easily follow droplets of these as they 
move across the perivitelline space before they completely 
disappear. Any point on the egg-surface where this dis- 
solution is taking place becomes the point of susceptibility 
at the instant of exposure to dilute sea-water or tap water. 
The most remarkable characteristic of this susceptibility 
during membrane-separation is its sharp localization. I 



have made observations on thousands of eggs and have yet 
to see an egg exposed during membrane-separation break in 
those regions from which the membrane is not lifting. This 
is especially well brought out by exposing eggs during the 
early stages of membrane-separation; for then only that 
part of the egg from which the membrane is just lifting, at or 
near sperm-entry, is susceptible to dilute sea-water. 
Similarly, any part of the egg from which the membrane has 
fully lifted is resistant, as is strikingly shown by exposing eggs 
just at the moment when the membrane is being lifted from 
the last point on the egg-surface. Then the break-down is 
only at this point; the zones from which the membrane is 
already off are resistant. When the membrane is fully off, 
the egg leaves the period of susceptibility. We may say, 
therefore, that a wave of resistance to dilute sea-water 
follows in the wake of the wave of susceptibility. There 
is an exceedingly rapid restitution-process in the ectoplasm 
following a momentary loss of resistance through the normal 
break-down of material in the ectoplasm which pushes off 
the vitelline membrane. 

If we attempt to come to some conclusion as to the mean- 
ing of this susceptibility, we must take into account the 
following facts: First, the resistance of the ectoplasm in 
those regions from which the membrane has not yet sepa- 
rated; second, the very rapid recovery of the ectoplasm 
after membrane separation; third, the apparent failure of 
the ectoplasm at the site of sperm-entry to show any greater 
susceptibility than the remainder of the egg-surface. In 
brief, we must keep in mind that this susceptibility is 
clearly localized; or, what is more correct, that the break 
in the egg which is the expression of decreased resistance 
to the hypotonic sea-water is only at the site, and in the 
moment, of membrane-separation. Now this does not 
mean that only at this point water enters the egg. Rather, 
it indicates that the point at which the membrane is sepa- 



rating is the point at which the ectoplasm is weakest in that 
moment. In the zone of membrane-separation it is dis- 
continuous and shows a break. The normal process of 
membrane-separation being due to a secretory process is 
bound up with water movement. As the ectoplasmic 
material goes into solution, it is washed away. In the 
normal process just enough water is present for this behav- 
ior. When the egg is placed in dilute sea-water, the picture 
is different; the normally occurring recovery in the zone of 
membrane-separation can not take place, and the ectoplasm 
breaks down further. Moreover, with the ectoplasm now 
gone, the endoplasm is without protection and complete 
cytolysis results. We are thus here dealing with a sensi- 
tivity due to actual progressive dissolution of colloids in the 
ectoplasm of the egg. 

The case of the egg of Arbacia is very interesting. Here 
differences in resistance to dilute sea-water shown by the 
unfertilized and by the fertilized egg at the time of mem- 
brane-separation are not so clear-cut. I was, however, 
able to prove that the period of membrane-separation also 
in these eggs is a critical one, by studying the swimming 
larvae reared from eggs treated with dilute sea-water before, 
during and after membrane-separation. 1 Fertilized Arbacia 
eggs exposed to tap or distilled water before or after mem- 
brane-separation and then returned to normal sea-water 
give rise to normal larvae. But eggs exposed to the tap or 
distilled water during the period of membrane-separation on 
return to normal sea-water show marked abnormalities in 
the late stages of gastrulation. There is here, therefore, 
definite evidence of a differential susceptibility which 
appears in clear-cut fashion; dilute sea-water exerts a 
decidedly deleterious action during the processof membrane- 

1 Just, rg2Sc. 



separation and the egg is thereby so profoundly altered 
that the normal processes of gastrulation are disturbed. 

The unfertilized eggs of Nereis limbata withstand treat- 
ment with tap or distilled water for three or four minutes 
before disintegration takes place. Beginning twenty-five 
minutes after fertilization, the eggs are even more resistant 
than the unfertilized. But during the twenty-five minute 
period immediately following the mixing of eggs and sperma- 
tozoa, the eggs show a very low resistance for they dis- 
integrate within ten to sixty seconds after exposure to tap 
or distilled water. The surface-changes described above 
for this egg run over this period of twenty-five minutes. 
Thus, the period of susceptibility to hypotonicity exactly 
coincides with the period of break-down of material in the 
ectoplasm — with release of the jelly-forming material — the 
amoeboid changes and the darkening of the egg. When the 
egg rounds up and clears, the period of lowered resistance 
to dilute sea-water passes off. 

These experimental findings on three forms — and I have 
used eggs of other forms as well — though they show varia- 
tions, nevertheless point to one conclusion: the period of 
ectoplasmic changes following fertilization is one of pro- 
found physical alteration. The changes that 1 have dis- 
cussed here which follow the attachment of the sperm-head 
to the egg-surface sweep over the egg as a wave of explosions 
that break down material in the ectoplasm. During this 
period of visible alteration of the egg-surface occur other 
measurable physico-chemical changes, as oxygen-consump- 
tion and heat-production. These we may consider briefly. 

The oxygen-consumption by eggs during various stages 
of development following insemination has been investi- 
gated by several workers. 1 Shearer 2 has reported his find- 

1 JFarburg, Loeb and JVasieneys, Meyerhof, Shearer. 

2 Shearer^ JQ22. 



ings on oxygen-consumption by the eggs of a sea-urchin 
during the first minute after their insemination. Says 
Shearer: "On addition of the sperm to the eggs there is an 
immediate consumption of oxygen. In the course of the 
first minute the uptake of oxygen is many times that of the 
same eggs one minute before the addition of the sperm, 
and more is usually taken up in the first minute than is 
taken up in the second and third minutes after the addition 
of the sperm taken together." Shearer thinks that this 
" great initial inrush of oxygen into the egg and a corre- 
sponding output of C0 2 within the first minute after the 
addition of the sperm" make it clear "that the sperma- 
tozoon sets up an instantaneous oxidation-process in the 
egg, which is unparalleled in the reactions of the animal-cell 
for its sudden character." 

This period of a great initial inrush of oxygen established 
by Shearer's findings coincides with the period of surface 
changes described above during which time also the egg is 
so susceptible to the action of dilute sea-water. During 
this period the colloids in the ectoplasm go into solution, 
material is destroyed. It is this solution at the sur- 
face of the egg that is accompanied by the great rise in 

Shearer has also investigated heat-production in fer- 
tilized eggs of sea-urchins. Rogers and Cole, 1 using meth- 
ods of higher precision, have reported findings on the 
heat-production by the eggs of the sea-urchin, Arbacia. 
Their results on the heat-production immediately following 
insemination are of most interest to us. These workers 
find that the rate of heat-production at the instant of 
insemination is ten to twelve times that of the unfertilized 
egg. Thereafter the rate of heat-production falls con- 
stantly for twenty minutes to 65 per cent, of the value at 

1 Rogers and Cole^ 1925. 



fertilization, remaining constant for the next thirty minutes 
to drop again by more than 10 per cent., remaining constant 
as far as the eight-cell stage. Farther than this the obser- 
vations were not carried. 

Rogers and Cole's figure gives the approximate rate of 

heat-production. In their words, ''the greatest period 
of heat-production occurs immediately upon fertiliza- 
tion/ 51 This period falls in with that of the surface 
changes described above. Say Rogers and Cole : "The 
fact that the greatest heat-production by the egg comes 
immediately after fertilization seems to us to make it plau- 
sible to say that the entrance of the spermatozoon induces 
a cortical oxidation-process, and that this process results in 
the elevation of the fertilization-membrane." On the basis 
of my observations and experiments, described above, these 
findings of Rogers and Cole can be more precisely explained. 
What they have measured is the heat liberated during the 
disintegration of the ectoplasmic colloids. 

In addition to increased oxygen consumption and heat- 
liberation other cellular processes can be related to these 
surface changes in eggs. 

The wave-like process of break-down in the ectoplasm 
by which the membrane is separated from the egg of Echina- 
rachnius described above, strikingly resembles the trans- 
mission of change in various tissues. The nerve fibre may 
be taken as an example because among animal cells it is the 
most highly excitable and the most rapidly conducting. 
As is well known, nerve when stimulated at one point trans- 
mits this local effect throughout its course. As the propa- 
gated effect^ the nerve impulse travels along the stretch of 
nerve, each point successively, beginning at that where the 
stimulus was applied, becomes electro-negative to all other 
regions of the nerve both behind and in advance of the 

1 Rogers and Cole, I.e. 



point. Thus an electro-negative wave sweeps along the 
nerve fibre as the impulse traverses it. 

During the transmission of the nerve-impulse there are 
no visible structural changes in the nerve. The propaga- 
tion of the effect of sperm-attachment over the surface of 
the egg, however, is clearly visible. Thus, in the egg of 
Arbacia a wave of turbidity followed by one of lightening 
marks its course. In the egg of Echinarachnhis it reveals 
itself: by the behavior of supernumerary spermatozoa that 
are in contact with an egg and are brought to a standstill 
progressively, the ones nearest the site of sperm-entry being 
first quieted; by progressive dissolution of ectoplasmic 
colloids; and by the break-down of pigment granules in the 
jelly that encloses the egg. 

Also the response of the ectoplasm during the period of 
membrane-separation to exposure to hypotonic sea-water 
reminds us of the action-current in a stimulated nerve. 

The high degree of susceptibility of the ectoplasm in the 
zone of membrane-separation and the resistance in the 
zones from which the membrane has not yet separated or 
from which it is completely lifted again suggest the electro- 
negative condition in the nerve fibre. 1 It may well be that 
this is more than a superficial resemblance; and for this 
reason these ectoplasmic responses would warrant further 

Whilst the egg passes through the period of surface 
changes under discussion, another property of the ectoplasm 
rises to sharp visibility, its contractility. Of the eggs 
described above, that of Nereis is the best in which to 
observe the shrinking and expansion following insemination. 
But this phenomenon can be followed in other eggs. Met- 
schnikoff years ago called attention to the contractile power 

1 Lillie, R. S., 1922. 



of eggs of sponges; 1 Bcrthold 2 clearly described it for the 
egg of the sea-urchin as an unmistakable consequence of 
sperm-contact with egg. A year later 0. and R. Hertwig 
suggested that eggs are endowed with contractility. 3 The 
earliest description known to me of contraction in the ecto- 
plasm is that given by Ransom in 1854 who very clearly 
described it as a consequence of insemination, a work which 
has not received the attention it merits. 4 

That contractility is inherent in the ectoplasm of eggs 
can be readily demonstrated. Unfertilized Arbacia-eggs : 
for example, may be drawn out into long tenuous strands 
by putting them among fibres of lens paper. Since 
in this egg when it is unfertilized the endoplasm is highly 
fluid, this elasticity is due to the ectoplasm (including the 
vitelline membrane). The normal contour is readily 
regained without loss of fertilization-capacity. Paramoecia 
likewise can squeeze themselves through fine tubes and 
assume most bizarre shapes, returning again to the normal 
appearance when the pressure is released. 5 Lillie, 6 speaking 
of the egg of Chaetopterus, says: "The protoplasm of this 
egg is much more fluid than that of any other egg I have 
tested; in consequence, the egg elongates even with rela- 
tively low centrifugal force." And later "The egg is an 
elastic sphere; it therefore elongates in the direction of 
centrifugal force." 7 I conclude from these statements that 
the elasticity of this egg is due to its surface. Mrs. 
Andrews 8 attributes elasticity of cells to the ectoplasm. 

1 Metschnikoff, 1S79. 

2 Berthold, I.e. 

3 Ilertzvig, 0. and R., 1SS7. 

4 Ransom, I.e. 

5 Just, 1928c. 

* Lillie, F. R., 190S. 
7 Ibid., p. 72. 

* Mrs. Andrezvs, 1S97. 



These manifestations definitely coincide with the break- 
down of material in the ectoplasm and with the rapid recon- 
stitution whereby the egg-surface builds itself anew. They 
are fundamental physiological expressions of vital activity 
and have their cause in the visible structural changes at the 
egg-surface which have been observed both on the normal 
egg and on that exposed to the action of dilute sea-water. 
Sperm-attachment is for the unfertilized egg the normal 
and best means of stimulation; as a stimulus the sperma- 
tozoon calls forth the same responses in the egg — oxygen- 
consumption, conduction and contraction — elicited by 
stimuli acting on other cells. This similarity I hold to be 
of fundamental significance for cell-physiology for I con- 
ceive these modes of response to be properties of the ecto- 
plasm of cells generally. The grounds for this conception 

Few investigators to-day support the old theory of the 
nucleus as the seat of cellular oxidation. The human red 
blood corpuscle is pre-eminantly an oxygen-carrier; it has 
no nucleus. If there be evidence of increased accumulation 
of oxygen in the region of the nucleus of a cell, this more 
likely is caused by the presence of the nuclear membrane 
rather than by nuclear substance. Positive evidence indi- 
cates that oxygen-consumption depends upon cell-struc- 
tures, that is, upon minute surfaces in the protoplasm; even 
triturated cells consume oxygen. 1 Then the cell surface 
constitutes, par excellence, an oxygen-uptaking structure. 
And since this surface is subdivided into processes and its 
area therefore increased many times, its effectiveness is 
enhanced. In higher organisms, as the vertebrates, the 
cells which take up oxygen are rich in surface — as cells of 
the gills in aquatic and of the lungs in terrestrial animals. 2 

1 Warburg, 1914. 

2 The entire inner surface of a man's lungs amounts to about 
90 square meters, more than TOO times the area of the skin. 



Red blood corpuscles are discs, measuring seven to eight by 
two microns; they thus possess a large surface area, the 
sum of which amounts in an adult man of 78 kilograms, 
according to one estimate, to 3840 square meters. Hart- 
ridge 1 has suggested that the shape of the red blood corpus- 
cle is the best possible one for insuring easy oxygen-intake. 

No one can object to the proposition that oxygen to enter 
a cell must cross the cell-boundary. Even on the theory 
that in oxygen-consumption only the most deeply located 
structures of the cell, nucleus or otherwise, are concerned, 
the oxygen enters the cell by way of the cell-surface. Rea- 
soning by analogy we may say that just as there exist special 
structures as gills and lungs to obtain oxygen, so in cells 
generally the modified surface permits easy access of oxygen. 
These surface-modifications are delicate prolongations. It 
is also interesting to note that pigment granules, said to be 
carriers of oxygen, line up at the cell surface. 

Oxidation means liberation of heat. The heat is lost 
by the cell-surface. Here again the cell-surface is admir- 
ably constructed for the rapid conduction and radiation of 
heat. Since heat is produced when colloids take up water 
and also when they liquefy, it may be that cell-surfaces in 
breakdown through swelling and liquefaction liberate heat. 

Another fundamental property of living substance is 
conduction. Any living cell when stimulated has the capa- 
city to transfer the effect of this stimulus. The degree of 
conduction constitutes one of the chief differences that dis- 
tinguish animals from plants. In animals conduction is 
the function of the nervous system, which possesses this 
property, general to all cells, in the highest degree. 

Now among other characteristics of nerve cells we note 
an extreme differentiation of the ectoplasm, as has been 
stated. Nerve cells show one or more processes, some of 

1 Hart ridge, IQ20. See also Ponder, 1934. 




which attain great length. Even the simplest unipolar nerve- 
cell, that is, a cell with one prolongation, is a cell in which 
the ectoplasm is drawn out into an extensive filament. 
The more richly branched cells and those with the longest 
prolongations show no cytoplasmic inclusions in the 
branches, i.e., the fibres; instead, the cytoplasmic inclusions 
are located in what is called the nerve cell body. Struc- 
turally, therefore, the nerve cell is not only rich in surface, 
but also in a special kind of ectoplasm. It is more than an 
accident that this structure appears in highly conducting 
tissues. The minute attenuated threads of nerve fibres 
are admirably adapted for transmitting impulses. 

I have already mentioned the work of Harrison who 
demonstrated that the growing nerve fibre shows definite 
surface changes at the growing tip. 1 This tip eventually 
becomes the means by which one nerve cell establishes con- 
tact with another, the junction being known as the synaptic 
membrane. The fibre from one nerve cell does not pierce 
the cell body of another, but simply comes into delicate 
contact with it; and so the impulse passes from one nerve 
fibre to another. Conduction thus is carried on primarily 
by the fibre. 

Conduction in nerve cells, in other words, being a peri- 
pheral phenomenon, is not unlike that in an egg cell, since 
the interior of the egg cell is not involved in the conduction 
of the fertilization-stimulus. In free living unicellular ani- 
mals one can not always clearly identify conduction as a 
surface phenomenon because often conduction is followed 
so closely by contraction that it is difficult to separate the 
two processes. Nevertheless, there are forms in which it 
can be shown that the conduction is purely a surface 


It is to be borne in mind that conduction by nerve cells 

in higher organisms is not restricted to one cell. In the 

1 Harrison, I.e. 



propagation of the nerve-impulse, for example, from a 
sensory ending which receives the stimulus to end-organ 
(e.g., muscle) which is affected, the impulse in traveling 
over afferent nerves to central nervous system and from the 
central nervous system to the efferent nerve, involves at 
least two and, more frequently, several nerve cells. The 
transfer of the impulse from nerve cell to nerve cell as well 
as the connection between the efferent nerve and the end- 
organ has been clearly demonstrated to be by means of 
fine fibrils. These, the prolongations of the nerve-fibre, 
are as clearly ectoplasmic as the fibres themselves. Thus, 
the integrative action of the nervous system is established 
by intercellular connections. I conceive all modes of inte- 
gration in the body of a complex organism, as the verte- 
brates and man, to be at basis dependent on intercellular — 
ectoplasmic — connections. 

The central nervous system integrates all systems of the 
body; these are at some distance from it and unlike it; the 
glands of internal secretion in animals which possess them, 
exercise even more remote control over one or another 
system. A more passive integration — and of a lower order, 
since it binds tissues only — is that by connective tissue. 
Finally, by means of intercellular connections, like cells, 
i.e., cells in the same sheet of tissue, are held together. 
These different modes of integration one can not think of as 
mutually exclusive; nor can one regard them as categori- 
cally distinct and separate from the, point of view of func- 
tion. Only the order or level of integration — of all systems 
of the organism, of some systems, of organs, of a tissue — is 
a special, distinct one in each case. So much has been said 
by authors and in so great detail concerning nerve- and 
hormone-integrations, that no time here need to be taken 
to discuss these types of integration. Suffice it to point 
out that nerve action influences the chemical output of the 
glands of internal secretion and that the secretions in turn 
may affect nerve-tissues; to an extent, therefore, they 



depend upon each other. Apparently, connective tissue 
exercises merely a passive restraint upon the subjoined 
tissues; it isolates and insulates. It is otherwise with the 
interdigitated cells in a given tissue. Their interdigita- 
tions constitute a means for bringing the cells directly into 

On the side of theoretical biology intercellular prolonga- 
tions deserve our interest. Hammar suggested that by 
them the unity of the multicellular organism may be main- 
tained, seeing in them a basis for some support of Whitman's 
idea of the inadequacy of the cell theory for development, 
on which I have commented above; here I wish to point 
out the following: 

A multicellular organism, as a human being, is a unit 
and acts as such through integration. The cellular con- 
nections of the first grade in importance are those of nerve. 
To secure unified action of the organism is a function of the 
nerve cells primarily. Alongside with nerve integration, 
we place that by the hormones; for they come from glands 
upon which life depends, without which the organism dies; 
these, except the pancreas, are derivatives of or include 
derivatives of the nerve tissue or that from which the nerve 
tissue is derived, namely, the ectoderm; these glands are 
the pituitary body, the thyroid apparatus and the adrenal 
body. 1 

This nerve integration, as that of the three important 
hormones named above, is remarkable because of the fact of 
the ectodermal origin of its carriers. As we shall see in 
a later chapter, ectoderm cells arise from those cells in 
the developing egg which are richest in ectoplasm. Hence 
the highest form of integration in the complex multicellular 

1 The internal secretion of the pancreas is derived from cells zvhic'h 
are neither nervous nor ectodermal in origin and therefore the pan- 
creas cells are an exception here. 



organism is by means of ectoplasmic processes formed by 
originally ectoplasm-rich cells and by chemicals furnished 
by originally ectoplasm-rich cells. 

Now the integration of cell with cell — the simplest mode 
of integration — -is by means of the ectoplasm. Thus it is 
evident that all forms of integration in the complex organ- 
ism are in the last analysis ectoplasmic. 

Contraction is like conduction a fundamental property 
of the living cell. The lowest forms of animal and plant 
life have capacity for contractility either permanently or 
at some time or other in their life-history. All the cells 
of a highly organized multicellular organism possess it in 
some degree. But this endowment may be reduced to such 
a low point that it is almost extinguished. With evolu- 
tion animals developed cells which were set apart as con- 
tractile tissues. These are the muscle cells in which 
this fundamental property of living substance is highly 
emphasized. Muscle cells are also characterized by having 
great length. The smooth muscle fibre is thicker than 
either type of the striated muscle. It is also the least 
responsive and shows the longest reaction-time. Theories 
of muscle-contraction that have the widest acceptance 
explain it as a surface-phenomenon. It is certainly true 
that contractile tissues have relatively large surface area. 
Their elasticity is undoubtedly a function of the surface — 
as in the case of egg-cells. 

The life-process of a cell is the sum and interaction of its 
manifold activities. The phenomena which together inhere 
in the state of being alive reveal themselves as activities of 
the protoplasm. No protoplasm exists without them. 
For respiration, conduction and contraction, the foregoing 
presentation offers proof that they are in particular mani- 
festations of ectoplasmic activity. That nutrition is like- 
wise bound up with the ectoplasm will become evident in 
the next chapter. 



IF ate?" 


pound in the inanimate world, and the most ubiquitous, 
holds the same place in the animate. Roughly, living cells 
can be said to be made up of two-thirds of water. Water 
is a structural part of every living protoplasmic system; life 
does not exist without it. Even when in a highly dessicated 
state, animals, such as rotifers and tardigrades, are not 
completely anhydrous. 

Living protoplasm is an aqueous colloidal solution. In 
view of the prevailing emphasis nowadays placed upon the 
colloid state of protoplasm, it is astonishing how little we 
know of the manner in which water forms a part of the 
living state. Is protoplasm an emulsoid colloid ? To what 
extent is it a suspensoid colloid ? Is it a structure in which 
water encloses other substances or one in which the other 
substances enclose water, or is it of both types? With 
these questions I refer to the ultra-microscopic structure of 
protoplasm — that is, of the ground-substance — for to this, 
as already pointed out, the colloid-chemist by his own 
definition of colloid-chemistry should address himself if 
he wants to study living substance, and not to the micro- 
scopically visible inclusions. The solution of many funda- 
mental problems in biology is postponed because of our 
ignorance of protoplasmic structure. The colloid-chemist 
could render great service to medicine and biology by dis- 
sipating this ignorance. As I see it, the main significance 
attached to the study of matter in that physical state called 
the colloidal is that it deals with solutions; and where it 



concerns watery solutions it may give not only information 
concerning the physical properties of particles which lie 
within a certain range of size but additional knowledge of 
the chemical action of water in a system of colloid particles. 
The study of watery solutions is especially valuable inas- 
much as protoplasm is such a solution. 

Water also is a functional component in the vital reac- 
tions which take place in the cell. It plays a leading role 
in the utilization of food materials: when these are broken 
up into simpler compounds by the process of hydrolytic 
cleavage water is taken up; when in turn these hydrolyzed 
cleavage products are synthesized, water is lost. Whether 
in a cell this cleavage or synthesis will occur, depends upon 
the presence of water. Thus the movement of water from 
place to place in a cell hangs together with the direction in 
which the reactions run. Cellular oxidations demand the 
presence of water. Water is an end-product of all energy 
changes. Both conduction and contraction in cells depend 
upon water. In the removal of secretory products and in 
the elimination of excretory, water is necessary. 

Water thus is both part of the protoplasmic structure 
and a functional component in vital manifestations. In 
other words, it is bound up with the two fundamental 
problems of cell biology, protoplasmic structure and func- 
tion. The rhythm of water loss and water gain which every 
cell exhibits during its history, serves by these fluctuations, 
by its transitory nature, to emphasize the significance of 
water — to offer us, as it were, a key to the puzzle of the 
oscillatory changes which characterize life. Although dur- 
ing its development the animal egg progressively loses water 
— a dehydration imposed upon the rhythmical water-loss 
and gain — it by no means ever reaches the stage of complete 
dehydration. Its changing water-content therefore indi- 
cates to us a possible approach to the problems, how water 



forms part of protoplasmic structure and how it plays a 
role in protoplasmic activities. Since the structure of the 
whole protoplasmic system is composed so largely of 
water, and its manifestations as a reacting system depend 
so greatly upon water, this structure must in some way 
dictate the distribution of water within the cell and hence 
determine the entrance and exit of water. 

When by chance I saw drops of water leaving an egg-cell, 
I was alert to seize the opportunity to follow by means of 
simple observations the fate of water within the egg. 1 
These observations will now be detailed and conclusions 
derived from them. Also suggestions will be made con- 
cerning the entrance of substances in solution into cells and 
concerning the question, why cells take in certain substances 
and not others. First, some mention should be made of 

When unfertilized eggs of marine animals are placed in 
dilute sea-water, they take up water and swell at a rate 
which depends, for specific eggs, upon the degree of dilu- 
tion. Thus, for the eggs of the sea-urchin, Arbacia, a 
favorite object for this type of work, the rate of swelling in 
40 per cent, sea-water (60 parts tap water plus 40 parts 
sea-water) has been measured by Lillie 2 and confirmed by 
others. After thirty minutes in this dilution the eggs 
break down. If the sea-water is still more dilute, the water- 
intake is more rapid and the eggs' break-down occurs earlier. 
If the sea-water is less dilute, the rate at which water enters 
is slower and break-down comes on later. Thus I found 
that in 60 per cent, sea-water Arbacia-eggs remain intact 
for twenty-four hours. Unfertilized eggs of Echinarachnius 
are more sensitive, being injured by dilutions which are 
innocuous to Arbacia-eggs. 

1 Just, 1926b, 1928b, 1930a and b. 

2 Lillie, R. S., 1916. 



According to Lillie, among any lot of unfertilized eggs of 
Arbacia which have been placed in 40 per cent, sea-water 
are some which disintegrate after a very short time whilst 
50 per cent, disintegrate after thirty minutes. From this 
observation it is clear that, since more than half of the eggs 
are killed by this dilution, the action of the 40 per cent, 
sea-water on them is not reversible. Also, it is questionable 
that this dilution is innocuous to those eggs that do not 
disintegrate during these thirty minutes ; they would 
scarcely return fully to normal condition when brought 
back to normal sea-water. Lillie points out that the eggs 
after eighteen minutes exposure continue to swell, i.e., fail 
to reach equilibrium with the diluted sea-water; instead 
they break down. Thus this dilution gives information 
concerning the rate of swelling only in injured eggs about to 
die. The very obvious question arises, namely, how much 
of the swelling is due to injury. It is thus difficult, if not 
impossible, to draw conclusions from these experiments 
concerning the behavior of normal eggs. Lucke and 
McCutcheon in their many calculations of the swelling of 
the Arbacia egg 1 have likewise used 40 per cent, sea-water 
as well as other solutions including non-electrolytes (sugars). 
But inasmuch as to their work the same objections hold as 
to that of Lillie — it is almost certain that the sugar solu- 
tions used injured the cells further — we can not here con- 
sider it, although, for their purpose, the demonstration of 
the osmometer-properties of the egg, its moribund condi- 
tion is of no consequence. 

In all my work I was at great pains to fix very definitely 
the lowest limit of hypotonicity that a given marine egg 
can withstand with recovery to the perfectly normal condi- 
tion. The study of dead protoplasm as it exudes from an 
egg membrane because of pressure put upon it either by 

1 Lucke and McCutcheon, J929 and earlier. 



mechanical means or by hypotonicity may be interesting 
and even fascinating and may allow the drawing of some 
conclusions concerning the normal processes in the living 
state. However, in the elucidation of the normal processes 
in the living state, observations and experiments on living 
eggs whose conditions are only slightly altered, are, as was 
said in a previous chapter, of higher value for drawing con- 
clusions than the study of dying or dead eggs. Aly studies, 
first made for another purpose, on several species of marine 
eggs of the rate at which the deleterious effects of hypo- 
tonic sea-water set in, led me to establish those hypotonic 
solutions in which the eggs did not break down though 
they remained in the solutions for hours; indeed, in these 
solutions these eggs remained intact as long as, or longer 
than, normal eggs in normal sea-water. We may for our 
purpose here at once dismiss those grades of hypotonicity 
which destroy the eggs. 1 

There are grades of hypotonicity which though allowing 
unfertilized eggs to survive for hours, do in time destroy 
them, as does normal sea-water which also finally is destruc- 
tive. Such solutions one must use with caution because 
what is essential in these studies is the most satisfactory 
demonstration that the action of the solution can be fully 
removed — i.e., that eggs after residence in it on return to 
normal sea-water are as normal as those which never have 
been in the solution. Usually one measures the size of the 
unfertilized eggs in sea-water after they are removed from 
the hypotonic medium; the return to normal size is taken as 
the criterion that the egg has recovered completely. This, 
I find, is not enough. I have pointed out that the great 

1 // was deemed necessary clearly to establish in this elaborate 
fashion that the phenomenon occurred in viable eggs because after 
the first communication had been given and whenever demonstrations 
zvere made, the question, are the eggs alive? was raised. 



advantage of using eggs instead of other cells, especially 
tissue-cells from a many-celled animal, lies in the fact that 
one has more and sharper criteria for determining that the 
cells are alive and normal. One very excellent criterion is 
the fertilizability of the egg. Eggs that after return from 
a hypotonic solution regain normal size, may sometimes 
not be capable any more of fertilization or of develop- 
ment. Or, as has happened in my studies, the hypotonic 
sea-water induces parthenogenetic development. Fertili- 
zation-capacity is then the best expression of normality. 
If on return to normal sea-water the eggs not only regain 
normal size but fertilize and develop as normally as eggs 
that never had been in hypotonic sea-water, we may con- 
clude that they have fully returned to normal condition. 
In the table appended figures are given to show how far 
eggs of Nereis recover after exposure to various degrees of 
hypotonicity as measured by the per cent, of cleavage and 
of swimming larvae. 

Table I. — Per Cent, of Cleavage and of Swimming Forms of Eggs 

Fertilized in Sea-water One Hour after Having Been Returned 

from Dilution of Sea-water, in Which They Had Been for 

One Hour* 

Per cent, of sea-water 

Per cent, of normal 


cent, of normal 



















* This table represents one experiment of 10 made for each dilution. In most of the 60 experiments 
the controls showed 100 per cent, cleavage and more than 90 per cent, normal trochophores. 

If one bars accidents and uses proper care to guard the 
eggs against overcrowding and high temperature, they 
develop as if never having been out of sea-water of normal 



But I went farther in insuring the viability of the eggs. 
I used dilutions of a degree which allowed the eggs to fer- 
tilize and develop whilst in the dilution. Naturally, this 
development in the more dilute sea-water is far from normal. 
Nevertheless, if fertilization and development proceed in a 
solution, that solution is not as destructive as one in which 
both egg and spermatozoon are killed. The table appended 
gives figures to show the per cent, of development of eggs 
fertilized in hypotonic sea-water of various grades in which 
they develop. 

Table II. — Per Cent, of Cleavage and of Swimming Forms of Eggs 

Fertilized in Various Dilutions of Sea-water, in Which They 

Remain During Development* 



. of swimming 

Per cent, of sea-water 

Per cent, of cleavage 




















* This table represents one experiment of 10 made for each dilution. In most of the 60 experiments 
the controls showed 100 per cent, cleavage and more than 90 per cent, normal trochophores. 

I spent a great deal of time in taking these precautions 
because I wished very clearly to establish that the eggs in 
which I observed drops of water were living cells capable of 
normal processes. Many times indeed I fertilized eggs 
immediately upon their return to normal sea-water — that 
is, during the process of their return to normal condition 
it was possible to initiate development in them; hence the 
drop-formation did not interfere with fertilization. Thus, 
these observations were made on viable cells; I can think 
of no source of error left unguarded. 

Now let us follow the process in detail. 1 In the hypotonic 

1 Although incidental observations had often been made earlier, 
the first systematic ones were made in 1925 on which the initial 
report published the next year ivas based, 



medium the eggs gradually swell so that finally their bulk 
greatly exceeds that of the normal egg. With this increase 
in volume, the yolk-spheres, so striking in the normal egg, 
seem to disappear, actually they have changed to an unusual 
degree, losing their refringency and their discrete character; 
indeed, they may actually merge. These changes in the 
yolk are worth noting. What happens is this: The indi- 

; ^'.ff. , -:^:^^.:':- v / 


Fig. 2oa. — Section of a fixed egg of Nereis after 45 minutes' exposure to 
4<Dper cent, sea-water. The egg is swollen, the ectoplasm thickened and opaque, 
the yolk spheres are in process of fusion. (Figs. 20a to 20^ drawn by Mr. L. A. 
Hansborough from author's preparations). 

vidual yolk-spheres increase in size with a liberation of 
fine drops of oil, thus becoming almost transparent in 
appearance. If exposure goes farther, the yolk-spheres 
fuse. Yolk-spheres in fixed normal eggs are homogeneously 
blackened by iron haematoxylin; the yolk of the swollen 
egg also stained with iron haematoxylin is seen to be made 
up of fine threads within a distinct membrane, each yolk- 
sphere thus resembling a nucleus. The germinal vesicle 


(nucleus in the resting stage) is also swollen. The ecto- 
plasm is so narrowed that its fibrillae seem absent; in other 

O °" V 



SL. '" 






Fig. 20&. — Section of egg, in the same stage as that of Fig. 20a, after having 
been returned to sea-water. The yolk spheres are regaining normal structure; 
water in drops moves toward the periphery. 

Fig. 20c. — Same history as that of Fig. 2oi, but a later stage of development. 

words, the pressure of the egg-contents has reduced the 
width of the ectoplasm. On return to normal sea-water, 
the egg shrinks and the water-drops then appear. The 




— O 


v - •" * I 






Fig. 20<i. — Same history as that of Fig. 2or, but a still later stage of development. 

Fig. zoc- Egg of Platynereis me galops showing water-drops in cells of a late 
cleavage-stage on return to sea-water following exposure to 40 per cent, sea -water 
for one hour. 



accompanying figure (Fig. 20) illustrates these changes as 
seen in fixed eggs. First the drops are at or near the egg- 
centre, then they move outward. As they cross the ecto- 
plasm they become elongated. Whilst the water-drops 
form and the egg regains normal shape, the yolk-spheres 
return to their original condition; they again become dis- 
tinct spheres in the living egg, appear blackened in the 
fixed. This change runs parallel with the movement of 
the fine oil droplets into the yolk. 

Thus the movement of water from the egg is associated 
with a marked behavior of the yolk-spheres. As the cells 
take up water, the yolk-spheres lose oil and increase in 
size, even fusing to make one mass. That is, the yolk- 
spheres take up water from the water-logged protoplasm. 
Then as the cells lose water to the surrounding medium, 
the yolk-spheres lose water to the cell and regain the oil 
from the cytoplasm. In the case of the Nereis egg the 
yolk-spheres possess a very exaggerated water-holding 

It would be a mistake, however, to conclude from this 
description that the yolk-spheres are the sole regulating 
mechanism for the water control in the cell. Water-drops 
also form in the nucleus, in the clear cytoplasm in cells of 
stages of late cleavage which contain no visible yolk-spheres, 
and likewise in the gut-cells of larvae after all yolk has dis- 
appeared. Other animal cells than eggs similarly I find 
show these drops of water. One factor which determines 
the strong expression of water-drop formation is doubtless 
the ectoplasm, because eggs and other cells that possess 
most pronounced differentiated ectoplasm show water-drop 
formation best. 

The rate at which the drops appear I have studied. A 
comparison of this rate in eggs in various stages is inter- 
esting: Drop-formation is more rapid in fertilized than in 
unfertilized eggs. It varies in fertilized eggs depending 



upon the stage in the division-cycle of the egg. The forma- 
tion of water-drops thus is also a cyclical phenomenon. 

From these experimental findings we may draw the fol- 
lowing conclusions concerning the behavior of water in 
these eggs. 

First, water leaves the cell as discrete drops. This does 
not imply that all the water that leaves the cell is in this 
form. But since, as is shown in these experiments, water 
leaves the cells in visible drops, a theory concerning the 
exit of solutions as ions can not apply to these drop-forma- 
tions. Visible drops of water are of more than even mole- 
cular size. 

Second, the change in shape of the drops as they cross 
the barrier of the ectoplasm suggests that they pass through 
canals whose diameters are less than theirs. Now the ecto- 
plasm shows chambers, the spaces between the radial pro- 
jections. Since the diameter of the drops is less than that 
of these ectoplasmic chambers, the latter can not be the 
canals concerned. The canals therefore through which 
the drops pass though not sub-microscopic are smaller than 
these ectoplasmic spaces. The experiments thus strongly 
indicate that under these conditions the ectoplasm is a 
sieve with extremely small openings. 

Third, the yolk-spheres take up water from the cyto- 
plasm. The yolk in the Nereis-egg is a mixture of lipoid 
and protein. 1 This combination breaks down as water 
accumulates in the cell and enters the yolk-spheres. Lipoid 
escapes from the yolk-spheres into the cytoplasm. When 
water leaves the cell, water also leaves the yolk and the 
lipoid again enters the yolk. In hydration and dehydra- 
tion of the yolk the lipoid moves out of and into it. Oil 
moves out of the yolk as water moves into the yolk-spheres 
leaving them skeins of protein. As water moves out, oil 

1 See Konopacki, 1929. 



moves in and the yolk-spheres assume their original physical 
appearance. These changes in the yolk-spheres show one 
very delicate mechanism by which cells hold water. 

Fourth, the yolk-spheres are not alone concerned in the 
water-holding capacity of the cell. The clear, apparently 
structureless, cytoplasm and the nucleus have the power 
of holding water and of losing it in the form of drops. 

It remains now to be decided how far we can apply these 
conclusions on the findings on eggs under experimental 
conditions to normal eggs in normal sea-water. Although 
these eggs of the experiment were viable and capable of 
recovery and complete development, they nevertheless 
were subjected to an experimental treatment. 

However, these experiments of mine described above 
were made with greatest care within limits definitely set; 
the processes described can not be attributed to extensive 
or drastic injury. There is thus less danger in making 
statements concerning them as explanation for normal 
conditions than in drawing conclusions concerning normal 
processes from experimental procedures which killed the 
eggs ; the former only exaggerate, the latter extinguish the 
process that we wish to explain. 

If the process of drop-formation of water observed in 
these experimentally treated eggs is only an exaggeration 
of a normal process, it should be possible, I thought, to 
observe it both with other methods of treatment and in the 
normal untreated egg. As a matter of fact, I have found 
that the drops form as the result of pressure, of exposure 
to ultra-violet light and after removing the eggs from normal 
sea-water at 5°C. to that at room temperature (i9°C). 
During exposure to hypertonic sea-water, the drops are 
beautifully shown especially by fertilized eggs during every 
stage of development and by cells in the young worm. In 
unfertilized eggs in hypertonic sea-water the drops are 
neither so numerous nor so easily visible. 



In the untreated normal egg, drop-formation presumably 
occurs rapidly and the drops arc neither so large nor so 
numerous as in experimental condition. Actually I was 
able to observe the appearance of drops in normal unfer- 
tilized eggs. They are much smaller and more evanescent 
than in the experimentally treated eggs. They appear 
more clearly in the fertilized than in the unfertilized egg 
and vary in rate of formation during the cleavage cycle. 
Drop-formation is thus also in the normal egg related to 
the physiological rhythmical changes coincident with the 

Since these findings indicate that the experimental means 
merely prolong and render more easily visible the more 
fleeting changes in the normal eggs, we may with more 
confidence use the experimental results for interpreting 
normal conditions and processes. The experiments have 
far-reaching significance for cell-biology in two respects: 
they permit conclusions concerning an important aspect 
of the problem of protoplasmic structure and concerning 
the question of the movement of water into and out of 

Although we have some information on the struc- 
ture of protoplasm, we are far from possessing adequate 
knowledge of it. What is known of the chemistry of 
the cell does not suffice to tell us whether protoplasm is an 
emulsion, a suspension, a foam or a combination of these. 
As said above, an advance is made the moment that the 
ground substance is recognized as the cytoplasm par excel- 
lence. This appears as a menstruum containing extremely 
minute bodies. Since water makes such a large part of 
protoplasmic structure, we ought to seek to learn how it 
forms part of this structure: whether it encloses the par- 
ticles or the particles enclose it; or, in the words of the 
physical chemist, whether water is in the external or dis- 
persing or in the internal or dispersed phase. On this 



question the experiments showing that eggs of Nereis lose 
water in the form of drops throw some light. 

If we think of the water as in the external phase, the 
effect of increasing the density of the medium — from hypo- 
tonic to normal sea-water or from normal sea-water to 
hypertonic — would be either to cause streams of water to 
form rather than drops, or to free the water generally and 
equally everywhere from the eggs, so that the water would 
not be visible as it moves out of the egg. Since however 
drops of water form and since water can not appear in water 
as drops, we must assume that the drops are pressed out of 
structures. We conclude, therefore, that the water which 
appears in the form of drops is that which was held by solid 
structures, i.e., was in the internal phase. The formation of 
drops in normal viable eggs points to the same conclusion. 

As we have seen, the egg on return from hypotonic to 
normal sea-water regains its normal equilibrium as it 
shrinks. In this process, inasmuch as drops of water leave 
the yolk-spheres we conclude that the yolk-spheres possess 
water-holding capacity and thus exercise function in the 
distribution of water in the cell under the condition of the 
experiment. Because it is not easy to discern yolk-free 
areas of the endoplasm in the unfertilized egg of Nereis, 
one can not so readily determine to what extent water-hold- 
ing power resides in the cytoplasm. In the various stages 
of development — cleavage, bias tula, gastrula, larva (tro- 
chophore) and young worm — as the yolk becomes segre- 
gated into certain blastomeres leaving others yolk-free, it 
becomes easy to learn that yolk-free blastomeres or those 
in which yolk is not in visible spheres also hold water in 
the form of drops. Therefore we conclude that the clear 
homogeneous cytoplasm holds water within its struc- 
ture which exists in a state of fine subdivision. Moreover, 
the nucleus reveals water present in drops; these exist apart 
from chromosomes. Since the water drops can be demon- 



strated by several experimental methods we may say that 
slight experimental modifications of the protoplasm cause 
movement of water out of the structures which hold it. 
The conclusion is that water as an integral part of the col- 
loid structure of protoplasm is in the internal phase. This 
does not mean that no water exists otherwise in living 
protoplasm. On the contrary, we can only account for 
the observed initial shrinkage of an egg on return from 
hypotonic to normal sea-water as due to the invisible escape 
of water which doubtless represents, in part at least, water 
held as in the external phase. 

Thus we have answered the first question raised: how is 
water held in protoplasm? 

I may point out that these experiments as such have 
some significance for medicine. The distribution of yolk 
differs with different eggs, for instance in that of Nereis 
and PlatynereiS) as I have said above. Likewise, during 
its development the yolk changes in location as has been 
shown. Undoubtedly some of the difference in water- 
holding capacity noted among eggs of several species or in 
the same egg at different stages is to be attributed to this 
difference with respect to yolk. What is true of yolk may 
be true of other bodies in the cytoplasm. In diseased con- 
ditions as oedema and nephritis in which tissues hold an 
excessive amount of water, the structure of the cell may be 
a very important factor in determining this abnormal water 

I turn now to the consideration of the question of the 
movement of water into and out of cells. This calls for a 
statement concerning the nature of the cell-surface. 

When this question of the passage of water and dissolved 
substances into and out of cells is raised, especially in physio- 
logical work, by the term, cell-surface, usually is meant a 
membrane. This membrane is then spoken of as semi-per- 
meable because it is said to take up certain substances and 



not others. Much of the work on which the theory of 
semi-permeability is based is derived from work on artificial 
inanimate membranes. Many investigators regard the 
cell membrane as a molecular film; others treat it as such 
in their physico-chemical and mathematical disquisitions. 
The chemical nature of the postulated semi-permeable 
membrane around cells has often been discussed. Depend- 
ing upon the one or another theory, it is considered protein, 
lipin, a mosaic of lipin and protein or a more complicated 
chemical structure. As was pointed out, to some the mem- 
brane is a dead, inert thing, a precipitation out of the proto- 
plasm, or merely a surface conditioned by the external 
medium, etc. For others the membrane is a living part 
of the cell-structure. 

In the above given descriptions of eggs, a vitelline mem- 
brane has been spoken of. This is built by the egg as it 
develops to maturity and is of measurable width. Beneath 
this vitelline membrane can be seen in many eggs a very 
delicate structure, appearing in optical section as an 
extremely thin line, the plasma-membrane, also built by 
the egg; it is visible and of measurable thickness and not a 
molecular film. The surface-structure of the cell is, how- 
ever, not to be thought of as comprising only this plasma^ 
membrane. The deformation of the water drops as they 
leave the egg takes place in the surface-structure below the 
plasma-membrane. This cell-structure is the ectoplasm. 
On these eggs as on so many others it displays amoeboid 
activity. Changes in a plasma-membrane and certainly 
those in a molecular film can not account for the rhythmical 
difference in permeability so often described and postu- 
lated, 1 for which, however, the ectoplasm, both in width 
and in activity, offers a sufficient basis. 

1 For examples of rhythmical changes, see Bayltss, 1915* 



In general, while it has been postulated that the differ- 
ences in permeability noted both in a specific egg during 
different stages of its development, as before and after 
fertilization, and in cells of the same type, e.g., red blood 
cells, but from different animals, are due to the membrane, 
and that the cytoplasms are the same, the actual and visible 
changes in the surface-layer of cells have been ignored. 
Despite the excellent observations made by Mrs. Andrews 1 
on the spinning activity of the ectoplasmic surface, which 
others have abundantly confirmed, this type of rhythmical 
surface-activity no one has sought to correlate with the 
postulated rhythmical changes in permeability. Though 
the measurable ectoplasmic layer of cells differs in width, 
the size of the layer has not entered into the calcula- 
tions made on the entrance of water. The cell is a 
bag of watery solution; and being capable of deformation 
with return to its normal contour and size, this property 
of elasticity is resident in the surface-cytoplasm. But the 
elastic property of the cell-surface has been deliberately 
ruled out in the mathematical calculations on the entrance 
of water into cells. On the theory that the difference in 
rate at which water enters a specific cell in different stages 
depends upon the permeability of the cell-surface, i.e., the 
plasma-membrane, we should expect a most exhaustive 
analysis of the structure and structural changes of that 
surface — certainly we should scarcely expect that in addi- 
tion to ignoring these the theory would actually discount 
the elasticity of the cell-surface. 

Vitelline membranes are often chitin or chitin-like sub- 
stance; it may be that generally they are protein; the 
plasma-membrane also may be protein, lipin or what else. 
These considerations lose in interest in view of the fact that 

1 Mrs. Andrews^ lSg6. 



1 that cell-structure which controls ingress and egress is the 
ectoplasm which in large part has chemically the same 
make-up as the remainder of the cytoplasm since endo- 
plasm and ectoplasm are one continuous, though differen- 
tiated system. 

The question which disturbs the physiologists who say 
that there must be a membrane in order to keep the cell- 
contents from flowing out and becoming miscible with the 
surrounding medium may be answered here. The cell- 
contents withstand outflow because the cell material is 
one continuous system. It is not like mercury which is 
now one mass and then many drops which flow together 
again; rather, protoplasm is a cohering though extremely 
thin dilute jelly-like solution, whose biological character is 
revealed by its strong regional differentiation. Excreted 
or secreted material, including colloids, gets out of a cell 
when it is no longer a living part of it. It is broken off and 
dispelled. Instances in support of this statement are not 
wanting. In egg cells we have seen that at fertilization the 
colloids in the surface break down and escape through the 
vitelline membrane. Material moves out of cells by 
secretion — that is, the cell breaks off part of itself and 
washes this part away. 

The largest question in the permeability problem relates 
to the entrance of substances into the cell. If we attempt 
to answer this question we do it on the basis that the cell- 
surface is living ectoplasm, continuous with the remainder 
of the protoplasm, and is made up of a brushwork of 

In part, the question how substances enter cells is bound 
up with another, namely, why some substances and not 
others get into cells. For example, in the gut and in the 
kidney salts or sugars when present in equimolecular con- 
centration do not cross the cell-surface in the same amount. 
These two cases constitute stock arguments of the vitalists 



against a strictly mechanistic point of view concerning the 
entrance of substances into cells. 

The normal living cell carries out certain reactions in 
which water plays a part; it, therefore, needs either to take 
in water or to get rid of it. Also in cyclical changes, as 
those of division, in which the nucleus breaks down 
and reforms, water moves back and forth between nucleus 
and cytoplasm as well as between yolk and ground-sub- 
stance. Whilst the cell-contents may vary from moment 
to moment with respect to water present, the water within 
a cell tends to maintain a certain level characteristic 
of that cell. This level varies with different cells. Thus, 
the water content of human cells differs: the enamel 
of the teeth, spermatozoa, and bone cells are poor in 
water while cells of the liver, kidney, intestine, etc., 
are rich in water. The level also varies in a given cell 
with its activity or stage in life-history. Thus, gland cells 
when actively secreting and when at rest show different 
levels. An egg-cell will show different levels at different 
stages of development. Finally, the level varies with 
changes in the surrounding medium. Every cell tends with 
respect to water to come into equilibrium with its surround- 
ings. The level at which this equilibrium establishes itself 
depends upon the cell's specific composition, stage in its 
life-history and its activity at a given time. 

Since the water level within the cell tends to remain con- 
stant under the changes brought about by reactions, water 
moves in or out in order to maintain this level. In a way 
the movement of water between cell and environment is 
comparable to the movement of oxygen; it is a diffusion- 
phenomenon for water moves to the region of most concen- 
trated material, i.e., from the region of more to that of less 

The air which human beings breathe is composed of 79 
per cent, nitrogen, 20.96 per cent, oxygen, less than one per 



cent, carbon-dioxide together with the rare gases, helium, 
argon, krypton, in traces. The permeability of the cells 
of the lung for these gases is an important property for 
without the entrance of them into the lungs and their 
subsequent conveyance to all parts of the body where oxy- 
gen is given up to each living cell, life would cease. The 
air breathed out contains the same 79 per cent, nitrogen, 
but only 16 per cent, oxygen and an increase of carbon- 
dioxide amounting to 4.38 per cent. All of us appreciate 
that the figures reveal how much oxygen the living cells use 
and need and how much carbon-dioxide they form and get 
rid of. 

The passage not only of water into and out of normal cells 
in normal condition, but also of substances in solution mav 
be compared to the entrance and exit of these gases. Some 
pass in and out again in equal, others in diminished, and 
still others in increased amounts. Without the entrance 
of food-stuffs and the exit of waste the cells would die. 
The phenomena of significance are the utilization of incom- 
ing materials by the cells and the excretion of effete. What 
has been said concerning water, therefore, may be said of 
material in solution as salts, sugars, amino-acids. These 
enter or leave the cells according to the level at which they 
are present in the cells. They form part of the protoplasmic 
structure and also take part in reactions going on within the 
protoplasmic boundaries. If their concentration in the 
cell is low, the cell retains them as they come in. In order 
to learn what substances a cell uses, we should not merely 
inquire what substances get into a cell, but also what of 
these remain in it. We may imagine that more of a given 
substance in solution in the surrounding medium passes 
into cells than remains in them. 1 

1 The mistake is often made of attempting to learn what can get 
into cells by too severe treatment of them, injuring them by subjecting 
them to solutions of too great concentration. 



If the cell-membrane were permeable only to water, none 
of us could live because the cells in our bodies would receive 
only water. The physiologist therefore has finally to postu- 
late a membrane whose temporary break-down allows sub- 
stances other than water to pass. 1 But I think that there 
is more to the problem than mere passage across an inert 
boundary. The complexity of the protoplasmic organiza- 
tion and the many reactions going on in the protoplasmic 
system must determine, in part surely, what substances 
coming in shall remain. What determines the ingress of 
substances is the cell-surface. By this is not meant a 
membrane, semi-permeable or otherwise, a dead or living 
molecular film, but the whole ectoplasmic structure with 
its innumerable filamentous prolongations of living active 

Every cell's ectoplasm is built up of such prolongations, 
as has been abundantly shown in the chapter on the ecto- 
plasm. The fact that this discernible, richly filamentous 
structure exists, taken alone suffices to render untenable 

1 Bayliss, IQ22, p. JI2; cc As a further case of absorption, at all 
events as it appears to me, the cell-membrane or plasma-membrane 
may be considered. This is not to be regarded as a fixed permanent 
structure, but as produced by deposition of cell-constituents which 
lower surface energy at the interface between protoplasm and sur- 
rounding medium. Thus, it changes with cell activity and is in 
equilibrium with the cell contents as they alter. Thus, there is no 
difficulty in the membrane becoming permeable in the active state 
of the cell to substances to which it is impermeable in rest. Moreover, 
zvhen a fresh protoplasmic surface is produced by mechanical action, 
a new membrane is naturally deposited on it. This is no doubt 
why large particles can be taken up in phagocytosis through a 
membrane zvhich does not permit even sodium chloride in solution 
to pass. The particles actually break the membrane, zvhich closes 
again behind them, in the same way as a needle can be passed through 
a soap film, without bursting it, zvhereas a gas, such as hydrogen, 
nearly insoluble in the soap solution, only passes zviih extreme 

J 4S 


theories based on a membrane of whatever hypothetical 
physico-chemical composition. The presence of the fila- 
ments means that the exposed cytoplasmic surface is 
enormously larger than if there were only a smooth 
membrane, and at the same time that it forms a sys- 
tem of capillary spaces. But this surface is not merely 
a static structure. The cytoplasmic processes which char- 
acterize the ectoplasm are indeed fine pseudopodia and as 
such display constant activity. By means of them the 
cell has phagocytic power, ingests fine particles. Phago- 
cytosis is* indeed an activity exhibited by all animal cells 
and not only by Amoebae, white blood cells and fixed tissue 
phagocytes as those in the vertebrate liver. 1 The moment 
that we appreciate the normal structure and behavior of 
the ectoplasm, the problem of the entrance of water and 
of solutions is placed in a new light. The theories of cell- 
permeability with their discrepancies and conflicts can with 
profit be abandoned. Since the ectoplasmic pseudopodia 
respond actively to the environment they regulate the 
exchange between cell and external medium. 

All these considerations and data indicate that the sur- 
face-cytoplasm can not be thought of as inert or apart from 
the living cell-substance. The ectoplasm is more than a 
barrier to stem the rising tide within the active cell- 
substance; it is more than a dam against the outside world. 
It is a living mobile part of the cell. It reacts upon and 
with the inner substance and in turn the inner substance 
reacts upon and with it. It is not only a series of mouths, 
gateways. The waves of protoplasmic activity rise to 
heights and shape the surface anew. Without, the environ- 
ment plays upon the ectoplasm and its delicate filaments 
as a player upon the strings of a harp, giving them new 
forms and calling forth new melodies. But these are too 
nice for the undiscriminating ear of man. 

1 Cf. Geddes, 1SS3. 


The Fertilizatio?i-process 


two processes: nutrition and reproduction. The first is 
concerned with the blindly egoistic struggle of the individual 
to preserve itself. The second relates to the altruistic 
struggle of organisms to perpetuate their kind. Despite 
their interdependence the processes of nutrition and repro- 
duction have different values for the organism. Without 
food or the apparatus for the utilization of food, the organ- 
ism dies; without the reproductive apparatus (as sys- 
tems, organs, tissues or cells) sexual organisms can still 
live. The reproductive (germ) cells are sharply set off 
from all other (somatic) cells; they have the special burden 
of the perpetuation of the species. The sex-cell is therefore 
a thing apart, a tenant housed by mortal somatic cells and 
like them mortal while the tenancy lasts. House and ten- 
ant have a common origin. Their separation constitutes 
the first of the series of those differentiations that mark 
the development of an individual animal. 

The adult animal is derived from one cell, the egg, which 
by the process of cell-division becomes a mass of cohering 
cells. They form the germ-layers; these give rise to the 
various organs (or systems of organs) which compose the 
complex adult individual. In the course of this develop- 
ment are set off from all the other cells which make up the 
body of the animal certain ones, the primordial germ-cells, 
whose function is to produce either eggs or spermatozoa in 
bisexual animals or both of these in monosexual or herm- 
aphroditic animals. Thus, the germ-cells, first differenti- 




ated from the somatic, are in their turn differentiated from 
each other. 

The primordial germ-cells pass through a period of mul- 
tiplication which ends with the production of many cells 
called ovogonia (in the female) and spermatogonia (in the 
male) whose nuclei still possess the somatic number of 
chromosomes, i.e., the number characteristic for the species. 
Ovogonia and spermatogonia without increase in their 
numbers are transformed into primary ovocytes and pri- 
mary spermatocytes respectively by the pairing of the 
chromosomes in each nucleus; thus the somatic number of 
chromosomes is ,c reduced ? " to one-half, the gametic or 
haploid number. Then follows the period of growth which 
is especially expressed in the egg. The maturation 
(meiotic) divisions succeed the period of growth and differ 
somewhat in the male and female sex-cells. 

In maturation usually each primary spermatocyte divides 
into two secondary spermatocytes and each of these again 
into two cells which are called spermatids. Hence a pri- 
mary spermatocyte gives rise to four spermatids. By a 
series of cytoplasmic changes together with nuclear con- 
densation the spermatids become spermatozoa. Only 
spermatozoa are capable of fertilizing eggs, whilst many 
eggs, as we shall see soon, can be fertilized before, after or 
in various stages of their maturation. 

The maturation (meiosis) of the egg occurs as follows: 
at the end of the growth-period two divisions of the primary 
ovocyte follow each other paralleling those in the primary 
spermatocyte but the cells so arising are markedly unequal 
in size. The small cells, the polar bodies, contain nuclei 
enclosed by a minimum of cytoplasm. Of the three ovo- 
tids (or four, if, as sometimes happens, the first polar body 
divides) from one primary ovocyte there is only one, the 
mature egg, capable of fertilization; the two (or three) polar 
bodies are abortive eggs. 



This brief account of the history of the germ-cells sub- 
sequent to their differentiation from the somatic cells 
teaches us that they pursue different courses. Without 
going into details, I call attention further to three facts of 
general significance for all that follows in my presentation 
of the problem of fertilization. The first is that there does 
not exist a single animal whose spermatozoa do not originate 
from an egg; the second, that no animal spermatozoon ever 
develops alone; and the third, that fertilization is not indis- 
pensable for the development of animal eggs, for, as we shall 
see in the chapter on parthenogenesis, many eggs develop 
normally or experimentally without the presence of sper- 
matozoa. These three facts obviously warrant the con- 
clusion that the egg carries the greater burden in fertilization. 
That the spermatozoon is itself derived from the egg is a 
fact to be emphasized. Even where it is indispensable for 
the egg's development, we can not in the light of its origin 
regard it as the perfect antipode of the egg. Whatever its 
highly specialized powers and functions, they rest upon 
derivatives of the egg-substance whence the spermatozoon 
came. That the spermatozoon never develops without the 
egg, whilst the egg can develop without the spermatozoon 
is another way of saying that the egg alone carries the bur- 
den of development. In these differences between the 
gametes we recognize where lie the powers not only of 
fertilization, but also of the whole course of the future 
development; the spermatozoon is a cell reduced to the 
minimum potency through its differentiation, the egg is of 
all cells known the most potent by virtue of its peculiar 
differentiation. Structurally, the great difference between 
spermatozoon and egg relates to the cytoplasm. Out of 
the egg's cytoplasm the future adult organism is differ- 
entiated. I propose in the following pages to prove that 
fertilization, the initial act in the differentiation of the egg, 
is likewise a cytoplasmic phenomenon. In order to reach 



this definition, I examine (i) the structure of the sperma- 
tozoon and of the egg when they normally come together, 
(2) the changes in both subsequent to union and (3) their 
behavior at the time of union. The present chapter 
embraces the discussion of (1) and (2), the succeeding 
chapter confines itself to (3). 

For the majority of multicellular animals, the coming 
together of the male and female germ-cells or gametes, 
spermatozoon and egg, guarantees the perpetuation of the 
species. This coming together of the gametes is fertiliza- 
tion in the widest sense, without which the spermatozoa of 
all and the eggs of most animals die. In this meaning 
fertilization marks the beginning of a life though of course 
both gametes are alive; it is the beginning of a new indi- 
vidual brought into being through the loss of individuality 
of each of the two cells which are co-partners in the process. 
If fertilization in all animals which exhibit it were to occur 
in the same mode and at the same time, our attack on the 
problem of fertilization Avould not be so difficult; but we 
'encounter many differences. Always in biology must we 
reckon with differences and seek to determine to what 
extent they are "incidentia" or " differentia." The com- 
plexity and diversity of animal structure and behavior are 
either "incidentia" and as such defy reduction to simple 
terms or, as "differentia," they mask some one common 
factor to which we can reduce them. That there is one 
feature common to all fertilization-processes we shall see 
after we have evaluated the differences in the structure of 
the gametes as well as those in the time and the mode of 
their union. 

The partners in the fertilization-process are as described 
above markedly different from each other in their develop- 
ment to the moment of their coming together. In one 
respect only are they similar: their nuclei contain each one- 
half the number of chromosomes characteristic of the adult 



organisms from which the eggs and spermatozoa come. 
In this bringing together of the haploid chromosome- 
garniture from the mother and the haploid from the father, 
fertilization not only insures the constancy of the species' 
characteristic chromosome-number, but also, since the 
chromosomes are concerned in heredity, it maintains equi- 
librium between paternal and maternal inheritance as far 
as this is determined by the chromosomes. This similarity 
of nuclear structure in egg and in spermatozoon has thus 
significance for the development that results from ferti- 
lization, but as a static, non-changing factor it can not be 
related to that radical and far-reaching change in the egg 
that we denominate fertilization. We, therefore, turn to 
the discussion of the other characteristics of the partners 
and shall find in them many differences. 

Animal spermatozoa exhibit great diversity in structure. 
In general, they may be classified as flagellated and non- 
flagellated. The larger number of species of animals have 
spermatozoa of the former class. Typically one such sper- 
matozoon is made up of a head (the nucleus) which may be 
spherical, bullet- or lance-shaped, to whose anterior end 
generally is fitted a cytoplasmic cap, the so-called per- 
foratorium, which may be very blunt, as in the starfish- 
spermatozoon, or more spike-like as in the Nereis- or the 
Cerebratulus-sptrmatozoon. The cytoplasm located behind 
the head is called the middle-piece; it varies greatly but 
often contains one or more granules of a lipoid nature. 
The tail or flagellum, the third cytoplasmic structure, is 
continuous with the cytoplasmic film enclosing the sperm- 
head and may be of very great length; it is the locomotor 
appendage of the spermatozoon. 

Non-flagellated spermatozoa may have the form of a 
truncated cone. Or they are more definitely amoeboid in 
form, as in crustacean spermatozoa such as the lobster's, 
crab's, etc. That is, instead of the blunt contracted form 

i5 T 


of the Ascaris-spermatozoon, those of these crustaceans 
present more slender projections resembling the finer pseu- 
dopodia of an amoeba. These spermatozoa are highly 
interesting because they are first inverted and explode at 
fertilization. 1 

Spermatozoa despite these great variations in structure 
have certain features in common: they possess very little 
cytoplasm although the volume of this may be greater than 
the nuclear volume. Compared to the egg, any species of 
spermatozoon is extremely minute. One fact already 
mentioned above is to be emphasized again: sperm-cells 
capable of fertilizing eggs are always fully matured — that 
is, they are always spermatozoa and never spermatocytes 
or spermatids. 

Whereas most, probably all, species of animal sperma- 
tozoa are motile, with few exceptions eggs are incapable of 
locomotion. Whilst, moreover, animal eggs vary in size 
from a few microns in diameter to several centimeters (e.g., 
eggs of cursorial birds), the smallest species of eggs are 
larger than the largest species of spermatozoa. This 
greater bulk of the egg is due to its cytoplasmic mass with 
inclusions, oil, yolk, mitochondria, etc. If these are sus- 
pended in the cytoplasm they may amount to about two- 
thirds of the egg-volume; if they are pendent to the cyto- 
plasm, as in birds' eggs, for example, they make up all but an 
extremely small fraction of the egg. No eggs, including 
the so-called transparent ones, are entirely yolk-free. In 
comparison with spermatozoa, animal eggs as a class at the 
time of fertilization contain more cytoplasm and are richer 
in reserve or food materials, yolk and oil. 

As was pointed out, eggs in contrast to spermatozoa can 
be fertilized before, during or after their maturation, 
depending upon species. We distinguish four classes of 

1 See especially Kolzoff, igio. 

i5 2 


eggs according to the stage in maturation in which they 
are fertilizable. 

Class I : The egg reaches the stage just prior to the matu- 
ration-divisions. This is the so-called germinal vesicle 
stage characterized by a large nucleus. It there comes to 
rest and dies unless fertilized. Mere contact of the sper- 
matozoon is sufficient to cause the break-down of the 
germinal vesicle and the initiation of the maturation divi- 
sions. Eggs of various worms, Polystomiim, Gyrodactylus, 
A scar is, Sagitta, Nereis, Platynereis, Thalassemia, Myzo- 
stoma, and of the clam, Mactra, are examples of this class. 

Class 2: Here belong eggs which will not fertilize as long 
as the germinal vesicle is intact. They develop as far as 
the stage of first maturation and there remain until death 
unless fertilized. The eggs of the sea-worms, Thysanosoon, 
Prostheceraeiis, Chaetopterus, Phascolosoma, and of the clams, 
Cumingia and My til us as well as eggs of snails and of the 
ascidians, Ciona and Phaltusia, are examples of this class. 

Class 3 : Some eggs finish the first maturation before they 
reach the stage in which they are capable of fertilization. 
They come to rest with the second maturation spindle 
formed, one polar body having been extruded. Here 
belong eggs of many vertebrates; likewise the egg of the 
worm-like chordate, Amphioxus, regarded by some zoolo- 
gists as the ancestral form of the vertebrates. 

Class 4: Eggs of sea-urchins constitute an example of 
this class in which fertilization is possible only after comple- 
tion of both maturation divisions. These are called fully 
matured eggs. 

It is here necessary to make a sharp distinction between 
maturation, referring to the phenomena of polar body 
formation, and physiological ripeness, referring to the 
capacity of the egg-cytoplasm for fertilization, which as 
this classification shows reaches its optimum at various 
stages in the maturation-process (Fig. 21). 



The egg of the starfish is interesting. Whilst other eggs, 
as we have seen, have a very sharply limited period during 
which fertilization is possible, the egg of this animal, whose 
optimum period for fertilization is just after the dissolu- 
tion of the germinal vesicle, is still capable of fertilization 
at later stages of maturation and for a time after complete 
maturation. But inasmuch as the fertilization-process 

c d 

Fig. 21. — Diagrams to illustrate the four fertilization classes (after Wilson). 

a, Class I; b, Class II; c, Class III; d, Class IV. 

either before or after maturation is below normal, this egg 
belongs to Class 2. 

It is evident that an explanation of fertilization must 
cover these four classes of eggs. A theory that covers 
the fertilization of the sea-urchin egg and not that of eggs 
fertilizable in the germinal vesicle stage and in subse- 
quent stages of maturation would demand a separate 
explanation for each of the other three classes. This in 
turn would mean that fertilization differs in different eggs — 



that, widespread though the phenomenon is, it would reveal 
no common factor. This point of view is often maintained 
for all animal biology, some workers going so far as to say 
that every animal and every egg is a law unto itself. How- 
ever, such a point of view does not properly envisage a 
phenomenon as widespread as fertilization. It is our pur- 
pose to look beyond differences, to seek a common factor. 
This can not be related to a particular stage in maturation, 
as our classification shows. It thus becomes necessary 
to describe briefly the process of fertilization as it occurs 
in a representative of each of the classes in order to learn 
if any feature is common to eggs of all classes. Said other- 
wise, having examined the structural make-up of the 
co-partners in the moment when they come together in the 
act of fertilization, we address ourselves to an exposition 
of the events that follow this coming together. At this 
point one general word concerning the way in which egg 
and spermatozoon meet may not be amiss. 

Eggs of animals, in which the sexes are separate, may be 
laid before the spermatozoa reach them. In such cases 
the eggs are shed into the sea — or fresh water — with or 
without copulation between male and female. In other 
cases, eggs are reached within the female's body by sperma- 
tozoa deposited within her genital tract; here copulation 
between the sexes is the rule. Eggs in these cases may be 
deposited at once to undergo development outside the ani- 
mal's body. Or they may remain within the female where 
they undergo development completely or in part. The fore- 
going is true also of normally hermaphroditic animals — i.e., 
those in which the sexes are united. It should be added 
that among these, eggs and spermatozoa produced by one 
individual may unite, so-called self-fertilization; sometimes 
this is brought about by the presence of some structure 
that prevents the access of spermatozoa from another indi- 
vidual to the eggs. In many hermaphroditic animals self- 



fertilization can not occur because eggs and spermatozoa 
are not ready for fertilization at the same time. The failure 
of self-fertilization among hermaphroditic animals is more 
often assumed than proved. 1 None of these modes by 
which egg and spermatozoon are brought together can be 
correlated with the stage in maturation at which the egg- 
cytoplasm is : 'ripe" for fertilization. Nor can we say 
that fertilization and development within the organism 
are peculiar to higher animals. Among sponges, for exam- 
ple, the lowest form of animals that produce eggs and 
spermatozoa, the eggs pass through early development in 
the parent organism. That tapeworms, members of the 
third lowest group of multicellular animals, are character- 
ized by their strong male copulatory organs, is a fact which 
discredits a popular notion that copulatory organs are 
found only among the highest animals. Thus, the mode 
by which the union of egg and spermatozoon is accomplished 
has no special significance for the events that follow this 

In the exposition of these events, which now is given for 
each class, we shall begin with contact of egg and sperma- 
tozoon and end with the first cleavage of the egg. These 
are common points. Between lie those events whose differ- 
ences call for evaluation. I request the reader to note them 
in order the better to appreciate the discussion of their sig- 
nificance. Named in order, these events are: the initial 
changes at the surface of the egg; the arising of two star- 
like formations, the asters, associated with sperm- or egg- 
nucleus (with or without a discrete body, the centriole, at 
the centre of each aster); the coming together of the egg- 
and sperm-nuclei (sometimes called pronuclei); and the 
formation of the cleavage-spindle. 

1 Cf. Just, 1914b, and earlier workers. 



The statement made in the chapter, Life and Experi- 
ment, that we lack many details concerning the happenings 
in normal biological processes, can be proved when the 
history of fertilization in the various examples given below 
is reviewed. Although I have endeavored to choose a 
representative for each class whose fertilization is best 
known, we shall see that in no one have all the steps been 
completely followed. Fertilization is far from being a 
sterile field of research; there is no single animal egg for 
which the events, from the moment of contact of egg and 
spermatozoon to first cleavage, have been so adequately 
described in closely set stages that we can say that we pos- 
sess full information of the chain of events as a continuous 
process. The description of fertilization in four eggs, that 
now follows, will give us merely the chief outlines of a pic- 
ture which we shall try to make more complete by adding 
lines and details from the process in other eggs. Only 
then shall we draw conclusions and enter upon the discus- 
sion of fertilization. 

I describe fertilization as it occurs in the egg of Nereis 
limbata found along the Atlantic shores of America; it 
represents eggs of Class I. 1 

The fertilization of the egg of this easily obtainable 
marine worm can be controlled since the eggs are discharged 
freely into the sea where they are immediately mixed with 
the spermatozoa; one needs merely to collect the mature 
males and females separately and then to place them 
together in pairs at timed intervals to obtain fertilized eggs 
in a series of as closely set stages as one desires. One 
draw-back exists, namely, that sexually mature animals 
can not be found throughout the summer months when the 

1 This account is based in large part on that given by Lillie, iqii 
and 19/2. 



animals breed. Like many other forms, Nereis exhibits a 
lunar periodicity in its breeding; behavior and is sexually 
mature only during the period from full to new moon of 

each lunar cycle from June to 
September (at Woods Hole, 

Mass.). 1 

Although "ripe" eggs of 
Nereis limbata are available 
only during this particular 
moon-phase, their abundance 
and the clock-like precision of 
their development make them 
ideal objects for observation 
and experiments on fertiliza- 
tion. The fertilization-pro- 
cess as seen in the living egg 
is as follows: 

When discharged or re- 
moved from the female the 
egg measures about 100 by 80 
microns. It reveals in optical 
section at the centre a large 
formation, the germinal 
vesicle. Around this are 

graphs of Nereis eggs in a suspension of 

Chinese ink in sea-water (after Lillie). greenish spheres, the yolk, 
a, before insemination; b, three minutes among which are larger re- 
after insemination. r . it i m i 

tnngent bodies, the oil drops. 
Beyond the area of yolk and oil is a rim, the ectoplasm, 
made up of coarse strands disposed in a somewhat radial 
fashion which extend to the clearly discerned vitelline mem- 
brane. The eggs die in this stage with germinal vesicle and 
ectoplasm intact, unless fertilized or experimentally treated 
by means of inducing parthenogenesis. 

Fig. 22. — Drawings from photo- 

1 Lillie and Just, 1913; Just, 191 4, 1929a. 



Within three minutes after the addition of a drop of 
active spermatozoa to the eggs, remarkable changes take 
place in each egg to which a spermatozoon has become 
attached : a jelly flows out of the ectoplasm; the dull slightly 
turbid ectoplasm in the unfertilized egg gives way to a 
shining space crossed by strands. These changes can best 
be observed under the microscope by adding spermatozoa 
to eggs in sea-water which contains fine particles of Chinese 
ink. The two photographs appended show living eggs 
before and after insemination (Fig. 22). 

c d 

Fig. 2^. — The fertilization-cone in the egg of Nereis (after Lillie). 

After the spermatozoon has become attached to the egg- 
membrane, the egg beneath the site of attachment forms 
a nipple-like projection, the cone, which shows itself well 
developed twelve minutes after the mixing of eggs and 
sperm, as can be noted in the Figs. 23a and b. (Com- 
pare these figures with those taken from the egg of Rhyn- 
chelm is, Fig. 24.) Fig. 25 (from a drawing) pictures 
a living egg in Chinese ink and sea-water fifteen minutes 
after spermatozoa had been added to it. One marks easily 
the germinal vesicle (too strongly drawn), the yolk spheres 
and larger oil drops in the endoplasm, the ectoplasmic 
strands, the entrance cone and the spermatozoon, plasma 
membrane, vitelline membrane, the jelly and the surround- 



ing ink which extends into the jelly hull where the sper- 
matozoon is located. 

The entrance-cone below the spermatozoon now gradually 
recedes and pulls the membrane along with it so that the 

Fig. 24. — The fertilization-cone and sperm-entry in the egg of Rhynchelmis 

(after Vejdovsky and Mrazek). 

spermatozoon lies in a depression. During these minutes 
the egg undergoes the irregular changes of form and darken- 
ing already described in the chapter on the general prop- 
erties of the ectoplasm. When it regains regular form and 
clears, twenty-five minutes after insemination, the cone is 



no longer visible in the living egg; the spermatozoon, seen 
with difficulty in the period during which these changes take 
place, becomes again visible after they pass over. About 
fifty minutes after insemination, the sperm-head disappears 
within the egg^ leaving the tail and middle-piece outside. 

Fig. 25. — Living egg of Nereis in a suspension of Chinese ink in sea-water, fifteen 
minutes after insemination (after Lillie). 

About five minutes later follows the extrusion of the first 
polar body. The second is extruded about fifteen minutes 
later. The egg cleaves into two unequal blastomeres one 
hour twenty-five minutes after insemination. 

Many details of the fertilization-process in the egg of 
Nereis can be observed only by fixing the eggs with a suit- 



able reagent which faithfully preserves the oil and yolk as 
well as the nuclear structures, cutting them into thin sec- 
tions (three or four microns thick) and coloring them with 
a dye which stains both cytoplasmic inclusions and chroma- 
tin material. 

In sections of eggs fixed after insemination, the following 
facts can be ascertained: The ectoplasmic breakdown has 
occurred. The germinal vesicle has broken down and the 
first maturation spindle has formed. To the fertilization- 
cone when fully formed the spermatozoon is fixed by the 
sharp anterior spike 5 the perforatorium, which traverses the 
perivitelline space whilst the head, middle-piece and tail 
remain external to the egg. The fertilization-cone as in 
the living egg projects beyond the egg-surface and later 

What one learns in addition to these details, verifying 
the observations on the living egg, concerns the change of 
the perforatorium of the spermatozoon and of the cone. 
As the spermatozoon enters deeper into the cone, further 
granules appear at its tip, whilst it itself gains in staining 
capacity. The cone is sharply marked off from the remain- 
der of the egg-cytoplasm; it is homogeneous in appearance, 
free from yolk and actually different in physical make-up. 

At about fifty minutes after insemination, the sperm- 
head disappears within the egg. The cone, as can be recog- 
nized by its staining and by its maintenance of form, acts as 
a solid body which sinks into the egg exerting tension upon 
the sperm-head which stretches like a ductile strand. Fi- 
nally the strand breaks at the surface of the egg so that the 
external portion remains outside. This is the middle-piece; 
hence, only the sperm-nucleus enters the egg of Nereis. 
Cone and attached sperm-head, acting as one complex, 
now revolve through an angle of 180 degrees. Soon there- 
after a star-shaped formation, the sperm-aster, with a 
minute granule, the centrosome or centriole, at its centre, 



appears in front of the sperm-head (now designated the 
sperm-nucleus). This minute granule arises from within 
the sperm-nucleus 1 (Fig. 26). As the sperm-nucleus 
advances toward the centre of the egg, the aster grows in 
size and it and the centrosome or centriole then divide; 
parallel with the growth of the aster the sperm-nucleus 

* b 

Fig. 26. — Sperm-nucleus within the egg of Nereis (after Lillie); a, with cone 
attached and b, separated from cone. 

enlarges, losing its staining power. In the meantime, the 
entrance-cone has disappeared. 

The egg-nucleus during the progress of these events has 
undergone changes. First, as germinal vesicle, it breaks 
down. The first maturation spindle forms near the centre 
of the egg and moves to the animal pole where the first 
polar body and, after the formation of the second matura- 
tion spindle, also the second is given off. The chromosomes 
remaining in the egg constitute the egg-nucleus. The union 
of egg- and sperm-nucleus follows and thus arises the first 

1 Just, 1933b, But cf. Lillie, rg/2. 



cleavage or zygote nucleus about which form two asters of 
unequal size. The larger of these which can be traced 
continuously is derived from the larger sperm-aster; 
the smaller, it is believed, though it can not be followed 
continuously, represents the smaller sperm-aster. Thus 
in the egg of Nereis both cleavage-asters are derived from 
the sperm-asters, if it be true that the smaller sperm- 
centrosome and -aster persist. Since the middle-piece 
remains outside of the egg, the cleavage-centres held to be 
genetically continuous with those of the sperm-nucleus 
can not be derived from the middle-piece. 

The history of fertilization in the egg of another marine 
worm, Chaetopterus pergamentaceus, as given by Mead, 1 
may be taken to represent eggs of Class 2 which reach the 
fertilizable stage after break-down of the germinal vesicle. 

These worms inhabit U-shaped tubes thirty to forty 
centimeters long, only the ends of which project above the 
mud. In the laboratory, these bizarre-looking animals, 
having been removed from their tubes, may be kept for 
some days in running sea-water. The sexes are readily 
distinguished: the eggs give the posterior segments of the 
female a pale yellow color, whilst the similar segments in 
the male which contain spermatozoa appear milky-white. 
It is best to use eggs and spermatozoa removed from ani- 
mals kept each in a separate container of gently flowing 
sea-water within twenty-four hours after the animals have 
been collected. 

When laid or removed from the animal, the eggs of 
Chaetopterus are in the germinal vesicle stage, but when they 
come into the sea-water the germinal vesicle breaks down, 
and the first maturation spindle forms near the centre of 
the egg. This moves to the animal pole of the egg and in 
this stage the egg remains until death unless fertilized or 

1 Mead, JS9S. 



treated by some means to stimulate parthenogenetic devel- 
opment. At fifteen minutes after removal from the animal, 
every egg shows the first maturation spindle, its chromo- 
somes at the metaphase firmly anchored by means of the 

oo oO 

o Oo<{ 



ov 6 osOo o0 o°oo6o 

Fig. 27. — For descriptive legend see page 166. > 

spindle's outer pole to the egg-periphery. At any time after 
dissolution of the germinal vesicle the egg is fertilizable. 
The egg before rupture of the germinal vesicle while in 
the ovary is an irregular pear-shaped body whose upper 
two-thirds are covered bv a delicate vitelline membrane 



under which lies the ectoplasm, showing one or two rows of 
granules. As the germinal vesicle breaks down, membrane 
and ectoplasm move over the vegetal pole and thus enclose 
the whole egg which has assumed an ellipsoid form. Usu- 
ally the spermatozoon enters the egg at the vegetal pole 

o o M> 

o ° ° 

w o o 
Oo_ ° O 

V^jOo^S^oSo o°°0 £» 

%<g OOo o o u Qo ooo 

Fig. 27. — Stages of sperm-penetration, egg of Mytilus (after Meves). 

after this has been covered by the moving ectoplasm. 
(Fig. 27 from the fertilization-process in the egg of Mytilus 
shows ectoplasmic changes after fertilization for this class 
of egg.) 

Entrance of more than one spermatozoon is rare. Within 
the egg, the spermatozoon after having moved some dis- 
tance shows an aster with two centrioles; these move apart, 



each carrying a separate aster. This configuration, the 
sperm-amphiaster, together with the sperm-nucleus comes to 
lie near the centre of the egg. During this change in loca- 
tion the small solidly staining sperm-nucleus becomes con- 
verted into a lightly staining larger mass. In other words, 
the compact mass of chromatin composing the sperm- 
nucleus is resolved into diffuse faintly staining threads. 

The exact origin of the sperm-centrosome (centriole) is 
not known. According to Mead, 1 

The behavior of the sperm-centrosomes is in harmony 
with Boveri's theory of fertilization, but is not necessarily 
a confirmation of it; for the karyokinetic activities which 
are revived upon the entrance of the sperm are those 
leading to the formation of the polar globules. The machin- 
ery for these mitotic divisions is already organized, and it 
is quite as likely that the stimulus which starts it going 
emanates from the sperm-nucleus as that it emanates 
from the sperm-centrosomes. 

The sperm-amphiaster develops into the cleavage-amphi- 

The eggs of Amphioxus — Class 3 — are laid toward eve- 
ning during the breeding season of this animal. If sexually 
mature animals are removed from the sea-sand, in which 
they live, to clean sea-water, they will deposit their eggs. 2 
Both Sobotta and Cerfontaine s confirm an old observation 
to the effect that these eggs develop best when shed into 
sea-water already containing spermatozoa; if they lie in 
sea-water before spermatozoa are added, they are suscep- 
tible to polyspermy and develop abnormally. The eggs 
form the first polar body while in the body of the female 

1 Mead, 1S9S. 

2 Lwoff, 1892, reported that he was able several times during the 
season at Naples to obtain eggs and spermatozoa shed in the labora- 
tory. On two occasions during 1929 I was able to induce animals 
to shed. 

3 Sobotta, I.e.; Cerfontaine, I.e. 



and when laid are in the stage of second maturation. 
They possess a strongly marked ectoplasm which remains 
wholly colorless after treatment with a dye which stains 
the yolk-spheres. This ectoplasm is made up of spheres 
lying among a fine net-work composed of radially projecting 

: :&V : f;V-Vyh ^W/v&V^" 


Fig. 28. — For descriptive legend see page 169. 

strands of the egg-plasma. (See figure in the chapter, 
The Ectoplasm, p. 99.) 

On entrance of the spermatozoon, which takes place at 
the vegetal pole, the ectoplasm breaks down; according to 
Cerfontaine it begins to go into solution at the site of sperm- 
entry. The ectoplasmic spheres seem to liquefy and 
become confluent. Both Sobotta and Hatschek affirm that 



the membrane separates with extreme rapidity. The lat- 
ter also says that separation begins at the point of sperm- 
entry, whilst according to Cerfontaine, actual lifting begins 
at the animal pole. 1 Appended figures (Fig. 28) of surface- 
changes are of the egg of Petromyzon, a member of this 

After sperm-entry the bulk of the spermatozoon is car- 
ried inward leaving a remnant at the entrance-point which 

k I 

Fig. 28. — Surface changes, egg of Petromyzon, during stages of sperm-penetration 

(after Calberla). 

is still visible as late as the four-cell stage. Two equal 
asters arise from the division of the single aster found in 
the vicinity of the spermatozoon. Whether or not this 
aster contains a centrosome is not certain. From an excel- 

1 Hatschek has also recorded some interesting observations con- 
cerning elasticity of the membrane. 




lent description of fertilization in the egg of Amphioxus 
this statement is taken: "Very close to the head of the 
spermatozoon one often sees a small intensely stained point, 
which could very well be the centrosome, but in view of the 
many yolk-granules in the neighborhood that have the same 
appearance, I do not dare to decide this." 1 When the egg- 
and sperm-nuclei unite, the sperm-centres continue as the 
cleavage centres. 

The eggs of sea-urchins, fertilizable only after complete 
maturation, represent the fourth class to be discussed. We 
may take the egg of Arbacia as an example. 

The normal, 2 living, unfertilized egg of Arbacia is of j6 
microns in diameter and bounded by a thin elastic mem- 
brane below which is the delicate ectoplasm. The nucleus 
in the living unfertilized egg appears as a clear space lying 
in any position with reference to the polar axis of the egg. 3 
On fertilization the vitelline membrane separates beginning 
at the point of sperm-entry where a nipple-like protrusion — 
the fertilization-cone — from the egg-surface forms to pull 
in the sperm-head. The surface of the egg seen under high 
power of the microscope appears delicately crenated because 

1 Sobotta, i8g7, p. 40. 

-Normal, living, unfertilized eggs of Arbacia are spheres of 
approximately the same size and specific gravity, each enclosed in a 
jelly hull. They possess bright red pigment grannies evenly dis- 
tributed and never clumped. Eggs that do not satisfy these criteria 
should be rejected for experiment especially if they color the sea-zuater 
in which they lie; such discharge indicates the presence of moribund 
or cytolyzing eggs. 

3 This axis is an imaginary line passing through the centre of the 
egg and the point at which the polar bodies are given off. In sea- 
urchins'* eggs the polar bodies are always given of at the point 
opposite a tube in the jelly-hull of the egg. Because in these eggs 
the polar bodies are usually lost before the eggs are shed, the location 
of the tube in the egg's jelly hull is important for determining the 
egg's polar axis. 



of thread-like projections of granule-free cytoplasm into 
the space between the vitelline membrane and the egg- 
plasma. Later by the anastomosis of the free ends of these 
threads a very thin sheath forms; the threads and their 
enclosing sheath constitute the hyaline plasma-layer. 
Fifteen minutes after fertilization (temperature of the sea- 
water around 2i°C.) the hyaline plasma-layer stands out 
very sharply even under low power of the microscope. 
During this same period the sperm-head evolves as the 
sperm-nucleus with attendant cytoplasmic changes. 

Although I have found it easy to follow the history of 
the sperm-head in the egg of Arbacia : I prefer at this point 
to base the following description on the egg of the flat sea- 
urchin or :c sand-dollar," Echinarachnius. This I do 
because the egg of Echinarachnius being larger and not so 
highly colored as that of Arbacia lends itself readily to 
exact observation in the living state. For both, except 
for minor variations, the process is the same. Because of 
the significance ascribed to the middle-piece of the sperma- 
tozoon in echinids by Boveri's theory of fertilization, it is 
necessary to take up the history of the middle-piece in 
some detail. 

During the stages of sperm-attachment and penetration, 
the middle-piece reveals the same structure and position 
found in the free-swimming spermatozoon. That is, it is 
closely fitted to the basal end of the sperm-head and shows 
prominently a bipartite granule. In fixed preparations 
treated with a dye, haematoxylin, the nucleus of the sperm- 
atozoon stains bluish gray; the middle-piece, the outer 
limits of which are continuous with the nuclear membrane, 
is also gray. The middle-piece granule is seen as a sharply 
defined black body, lying — as in the free-swimming sperma- 
tozoon — in various positions in the middle-piece. Though 
in some views it shows up as a continuous horse-shoe shaped 
body and in others as two rods, it is in reality a body com- 




posed of two curved rods joined together by a delicate but 
clearly revealed bridge. 

Once the spermatozoon together with the middle-piece 
is within the egg — the tail does not enter — it rotates through 
an angle of 180 degrees. This rotation may, however, 
take place while the spermatozoon is still in the entrance- 
cone. At this stage occur the following changes: the 
sperm-head may reveal a more sharply stained outline, thus 
giving the appearance of a hollow tube; the outline of the 
middle-piece is no longer distinguished; and the middle- 
piece granule is closely stuck to the base of the sperm-head. 

After rotation, the sperm-nucleus is directed toward the 
centre of the egg. The middle-piece granule, hitherto a 
closely fitting cap over the base of the sperm-head, slips off 
carrying with it a thread of sperm-substance. Or it may 
be that the sperm-head swells except in the region at which 
the middle-piece granule is clamped to its base. Figure 
29a shows a sperm-head at this stage. Certainly the 
sperm-head now shows well defined difference in volume 
and form; these changes take place with the separation of 
the middle-piece granule. 1 

The sperm-aster, never found before the stage at which 
the middle-piece granule draws away from the sperm-head, 
is now well defined and distinct. Its spherical central 
portion, the astrosphere, appears in fixed sections as clear, 
homogeneous substance which does not stain. The astral 
rays are paths of clear granule-free ground-substance lying 
between rows of the cytoplasmic constituents — mitochon- 
dria, yolk spheres and oil drops. The astral configuration 
noted in properly fixed eggs bears the closest resemblance 
to that observed in the living egg. 

1 These changes in the sperm-head are not unlike those found in 
free-swimming spermatozoa fixed after agglutination with specific 
egg-sea-water that zvill later be discussed. 



Within the astrosphere the middle-piece granule is bril- 
liantly revealed — a black eccentrically placed body, in 
sharp contrast to the clear substance immediately around 
it and the grayish blue cytoplasmic constituents farther 
beyond. The granule has never been found at the centre 
of the astrosphere. This is easily established in those 
stages, like Fig. 29^, in which the astrosphere is well 

During the ensuing stages of sperm-penetration up to 
the stage of apposition of the nuclei, the sperm-nucleus 
increases in size. The greater the distance, and therefore 
the time, which the sperm-nucleus must travel before reach- 
ing the egg-nucleus, the greater is its increase in size. This 
distance, of course, depends upon the site of sperm entry, 
which may be at any point on the egg-surface, with refer- 
ence to the location of the egg-nucleus, which has no fixed 
position. It is therefore very easy to obtain a large number 
of stages during penetration. 

During these stages I have found a behavior of the mid- 
dle-piece granule which differs from that described by 
Meves for the egg of Parechinus — a difference which may 
be due to technique, though I doubt that since the his- 
tory of the granule in fertilized eggs of Arbacia fixed as 
those of Echinarachnius is like that of Parechinus. This 
difference is that the middle-piece granule in the egg of 
Echinarachnius separates from each of its free limbs a more 
minute granule. Thus each limb of the original bipartite 
granule in turn becomes bipartite. These two new forma- 
tions move away from the parent granule, each maintaining 
connection by means of a delicate thread. Figs. (29^), 
(29c) and show this formation. 

As the sperm-nucleus increases in size, its basal thread, 
at the tip of which is the middle-piece granule, becomes 
longer, an indication of the ductility of the sperm-nucleus 
during these stages. In form, therefore, it resembles a 




B £ 



« o* w 


* # if r y 

ft : 

i^a^;*^^!.- * j>*» ~?_v*$ 






o 6 c 


To . -^ 



•^ * 

Fig. 29. — For descriptive legend see page 175. 




- X 



: • 

* < 




* •- 

* ' i 


t ~ , 

*•- * » 


> i ♦ . * 



* * 

* ? 

<*V* *, 

v / 

v 0' 


* • 
,> > .1 - > t 


* • * > 


• ■ , * ' * . 

" * 


:?%** » 

V^n- » * ^tk C '*' r V^' —^die-piece 


Yi Gt 2 g. — History of the middle-piece of the spermatozoon within the egg, 
Echinarachnius par ma. (Original.) 



round or oval flask with a long neck. In earlier stages this 
neck appears solid; in later stages it is really a tube, as 
Figs. 29^ and 29*? show. 

Now in this stage the structure of the middle-piece gran- 
ule and its relationship to the sperm-nucleus are most 
clearly discerned. The sperm-nucleus and the middle-piece 
granule form one continuous complex. The tip of the tube 
arising from the base of the sperm-head spreads out slightly 
as very delicate threads to connect with the minute granule 
which arose from the middle-piece body. Thus, the middle- 
piece granule resembles the top of a parachute, the threads 
of which come together at the tip of the sperm-tube. At 
this junction another granule is often found. 

With the apposition of the nuclei, the sperm-nucleus 
loses connection with the middle-piece granule. This is 
shown in Figs. 29/ and g. There is never a re-establish- 
ment; in the succeeding stages in mitosis leading to first 
cleavage, the granule may lie as a discrete single inert body 
at any point in the cytoplasm with reference to the spindle. 
I have never found any evidence of its division or of its 
taking up a position at either spindle pole. By the time 
that the nuclei come into apposition, the more minute 
granules can no longer be traced. 

Within the egg the sperm-head, a structure notable for 
its low water-content, at first maintains its shape and size 
but as it is carried from the periphery of the egg it slowly 
approaches spherical form, increasing in volume and its 
outline losing definition. Observations on eggs fixed during 
this period reveal what the living egg does not so clearly 
show: namely, that the transformation of the sperm-head 
into a vesicular nucleus is the resolution of a greatly con- 
densed mass of chromatin — an exaggerated and rapid evolu- 
tion of chromosomes from telophase condition to that of a 
resting nucleus. Though this process is more easily visible 
before the sperm-nucleus unites with that of the egg^ never- 



theless it can be discerned after this union. Finally, the 
sperm-nucleus loses its visible identity through complete 
fusion with the egg-nucleus. Soon thereafter two asters 
arise, presumably from the single sperm-aster. 

The foregoing accounts of fertilization, embracing eggs 
of the four classes made with respect to the period in matu- 
ration when eggs are in the stage for reception of sperma- 
tozoa, reveal two phenomena as common to all animal eggs. 
First, after attachment of the spermatozoon to the egg- 
surface, the egg-surface undergoes a change with the result 



d e f 

Fig. 30. — Surface changes in the egg of Mitrocoma attending sperm-entry (after 


that the vitelline membrane becomes separated. The sur- 
face-changes differ in quality : in eggs of Nereis a superficially 
located jelly is extruded; in the Chaetopterus-egg the obser- 
vable changes are less striking; in the egg of Amphioxus 
discrete bodies flow together and liquefy before the mem- 
brane separates widely from the egg; in sea-urchins' eggs 
the briefly enduring surface-changes are most violent. In 
order further to elucidate such surface-changes, I include 
pictures of a Medusa-egg (Fig. 30) described by Met- 
schnikoff. 1 It should also be mentioned that the entrance- 

a/ yo 




Metschnikojf, 1SS6. 


•n t . . .• * 

1 fa» v 

^" > .• - % 


cones may be exaggerated in unripe echinoderm eggs, i.e.. 
those incapable of fertilization and development (Fig. 31), 

Fig. 31. — Ectoplasmic changes in unripe eggs in response to sperm-entry, a, egg 
of a starfish (after Schneider); b, egg of a holothurian (after IwanzofT). 

The cones may be made more expressed under experimental 
conditions. 1 ]n the second place, the sperm-nucleus having 
reached the egg-nucleus forms with it the zygote- or 

1 Fol, 1S79; Schneider, 1S93; Iwanzoff, iSgS; Just, /929b. 



cleavage-nucleus, about which the first mitotic figure arises. 
Thus the fertilization-process in these four examples resolves 
itself into two phases- — an external, that concerns the ecto- 
plasm, and an internal, that concerns the nuclei. 

Fertilization means in the strict sense of the word the 
coming together of egg and spermatozoon, for without it 
there is no fertilization. Hence obviously, the initial act 
in the whole chain of events is the contact between the 
two partners. If we endeavor to state the definite result 
of these events, we can say that it is the development of 
the egg. But it would certainly be impractical to define 
fertilization as some have defined it, namely, that it is 
complete only when the developed organism has reached 
that stage in its development when in its germ-cells the 
chromosomes pair. Since in all animal eggs a mitotic figure 
is established for the first division-nucleus, it is far more 
practicable to take this feature, common to all eggs after 
development has begun, as the end-point of the series of 
events that begins with the coming together of egg and 

These events we speak of as the fertilization-process; 
this resolves itself, as said above, into an external and an 
internal phase. I shall now endeavor to determine which 
of these must be regarded as the fundamental phase in fer- 
tilization, that is, during which takes place the funda- 
mental happening, event, in fertilization. I first discuss 
the internal phase of the fertilization-process, since it 
involves more generally known phenomena and since pre- 
vailing opinion among biologists places the basic event in 
this phase. I begin with the question of the union of the 
egg- and sperm-nuclei. 

Were Ave to conclude from the descriptions of fertiliza- 
tion of eggs of the four classes as given above, we could say 
that fertilization is the union of the egg- and sperm-nuclei. 
However, cases exist in which cgg^ and sperm-nuclei norm- 



ally never fuse, although the presence of the sperm-nucleus 
within the egg is necessary for the egg's development. 
Indeed, in extreme cases the cleavage-nucleus is distinctly 
bipartite and remains so during many successive cleavage- 
stages. In the egg of the water-flea, Cyclops, for example, 
according to Haecker such a bipartite nucleus persists from 
one generation to the next. 1 Bipartite nuclei have been 
described for other eggs- — it shows clearly in those of the 
large tailed amphibian, Cryptobranchus. The egg of 
Pediadopsis presents an interesting condition : some of 
the cells resulting from its first division after fertilization 
contain single nuclei whilst in others each chromosome per- 
sists as a separate entity with its own spindle. Between 
the two extremes of complete fusion at the time when the 
egg- and sperm-nuclei appose and the persistence of these 
nuclei as revealed by the bipartite character of the cleavage- 
nuclei, intermediate grades exist, even in one egg-genus. 2 
In eggs of Rhabditis, though they must be fertilized in order 
to develop, the sperm-nucleus remains inert and never 
unites with that of the egg. 3 Briefly, actual fusion with 
immediate loss of identity of the egg- and sperm-nuclei is no 
sine qua non of the fertilization-process. 

Experimentally, it can be shown that fertilization may 
take place without the egg-nucleus. The presence of 
either the egg- or sperm-nucleus alone suffices for the 
egg's development: the former is concerned in parthe- 
nogenesis, the latter in an egg whose nucleus has been 
actually or virtually removed by experimental means. The 
fertilization of an egg-fragment without the egg-nucleus 
is known as merogony. 4 Such fertilizable fragments have 
been obtained from eggs of sea-urchins, starfishes and 

1 Haecker, iSpo. See also Ileberer for literature. 

2 Cf. Boveri's observations on eggs of the genus, Echinus. 

3 Kriiger, /pJ 'j. 

4 Delage and others. 



worms. Fertilization of eggs whose nuclei are hindered 
from taking part in development has been accomplished on 
eggs of sea-urchins and of amphibia. Experimentally it is 
thus shown that neither fusion nor even mere apposition of 
the sperm- with the egg-nucleus is essential for the fertiliza- 

Some observations of my own bear on this point. The 
first of these shows that in eggs of Echinarachnius , fertilized 
after having been treated with dilute sea-water — or after 
having lain in sea-water for several hours — the egg-nucleus 
takes no part in the subsequent cleavages but remains as a 
"resting nucleus" in every way similar to its condition in 
the unfertilized egg. 1 In the meantime, the sperm-nucleus 
divides in the typical manner of the zygote-nucleus. The 
second observation relates to eggs of the sea-urchin, Arbacia, 
fertilized in various stages of mitosis induced by treatment 
with hypertonic sea-water. 2 In some of these eggs the 
egg- and sperm-nuclei divide independently though in dif- 
ferent tempo, as one would expect. Here then the stage 
in the mitotic activity of the egg-nucleus does not inhibit 
the independent activity of the sperm-nucleus. 

The evidence derived from observations on normal fer- 
tilization-processes indicates that fertilization can occur 
without fusion, union, apposition, or even approach of 
the sperm- and egg-nuclei. The experiments cited show 
that development ensues when the egg-nucleus is absent or 
when though present it is rendered inert. We are therefore 
not justified in retaining the old definition of fertilization 
as fusion of the egg- and sperm-nuclei. 

To speak of the Rhabditis-egg, for which sperm-penetra- 
tion is essential to development, as "nature's bridge 
between parthenogenesis and fertilization/' 3 because the 

1 Just, 1924. 

2 Just, 1922b. 

3 Bracket, 191 7. 



sperm-nucleus once in this egg lies inert whilst the egg- 
nucleus alone takes part in development, is a pretty state- 
ment without much scientific value. If an egg can not 
develop without fertilization, the criterion of its having 
been fertilized is its development. If the spermatozoon 
is essential to the initiation of development, fertilization has 
been effected, if this egg develops. To speak of this case 
and of experimental conditions in which the egg-nucleus 
though present is inhibited from taking part in cleavage as 
partial fertilization is to ignore the essential problem. If 
an egg-fragment without a nucleus develops after having 
been entered by a spermatozoon, it has been fertilized. To 
retain the old definition of fertilization as the union of egg- 
and sperm-nucleus is to violate both fact and logic. 

But whatever the situation with respect to a union of the 
egg- and sperm-nuclei, always a division-spindle arises and 
in this the fertilization-process reaches its culmination. At 
the poles of this spindle are star-like formations, the asters. 
These beautiful formations are especially striking in many 
living eggs and since they are always present in the nuclear 
division of cleaving fertilized eggs, they have been held to be 
the cause of fertilization. Thus Boveri postulated a theory 
of fertilization which, though he himself later abandoned it, 
is even to-day defended by many writers: namely, that the 
essential feature in fertilization is the introduction into the 
egg of two centrosomes by the spermatozoon, about which 
two asters form which persist as the asters of the egg's divi- 
sion-spindle. Were it not for the fact that many writers 
still uphold this definition, we could dismiss it at once inas- 
much as we have seen in the descriptions of the fertilization- 
process of the four types of eggs given above that the cleav- 
age-centres can not be always shown to have arisen from 
sperm-centres. Moreover, whilst in the majority of eggs the 
cleavage-centres probably are continuations of both sperm- 
centres, there are eggs in which the centres come one from 
the egg-nucleus and one from the sperm-nucleus, as in eggs 



of many snails. In addition, there are eggs in which the 
asters of the division-spindle arise entirely around the egg- 
nucleus, the sperm-nucleus never showing any trace of 
astral radiations, as in eggs of trematodes. Finally, as we 
have seen, the division-spindle in eggs of sea-urchins shows 
asters devoid of centrosomes. What in these Boveri iden- 
tified as centrosomes have been proved beyond doubt to 
be bodies located in the middle-piece of the spermatozoon 
that are cast off into the egg-cytoplasm and have no causal 
relation to the asters. 1 For other eggs it has also been 
shown that granules in the middle-piece of the sperma- 
tozoon can not be identified as centrosomes. 2 In one egg 
only, that of Nereis, has it been shown that the centrosomes 
arise out of the sperm-nucleus. 

The conclusion is patent: as it is the case with the stage 
in maturation when eggs reach the fertilizable condition, 
and with the mode of union of the egg- and sperm-nuclei, 
so with the origin of the cleavage-centres — all possible varia- 
tions exist. Hence we find no constancy with respect to 
the origin of the cleavage-centres and therefore can not 
define fertilization as the importation into the egg of cen- 
trosomes by the spermatozoon. 

Failing to discover in any of the events that happen dur- 
ing the internal phase of the fertilization-process the funda- 
mental act in fertilization, we turn to the external phase, 
to the changes at the egg-surface incident to the attach- 
ment of the spermatozoon. All animal eggs in response 
to sperm-attachment show some visible change in the ecto- 
plasm. In the following chapter I present my hypothesis 
that underlying these changes at the egg-surface is the 
fundamental act in the fertilization-process. I denominate 
this act the fertilization-reaction. 

1 See Aleves, 1912; Just, 1927a. 

2 According to Aleves there are not two but five such granules in the 
sperm-middle-piece of Aiytilus. 

is 3 

The Fertilizatio?i-reactio?i 

It is a curious fact that by far the largest body of 
data, both of observation and of experiment, on fertiliza- 
tion relates to the end results and final consequences of the 
coming together of eggs and spermatozoa. The phenomena 
embracing the union of egg- and sperm-nuclei and the estab- 
lishment of the first cleavage-figure are end-events of a 
chain of happenings in a complex, heterogeneous and little 
understood system and not the end-point of a single one- 
way reaction taking place in a simple, homogeneous, fully 
understood system. Many biological processes doubtless 
may be explained on the assumption of an underlying sim- 
ple, even mono-molecular, reaction so far as such in chemical 
experiments with pure reactants be known or postulated. 
Nevertheless, to ignore the polyphasic nature of the cyto- 
plasm needlessly obscures the problem; comparisons of 
cytoplasmic processes with simple or even with complex 
reactions in test-tubes may cause serious retardation in 
the solution of the problems of biological behavior. Chem- 
istry, in so far as it relates to end-points, offers little help. 
As in chemistry more information regarding the onset of 
even the simplest reactions is desired, so here in the problem 
of fertilization: we stand in need of more exact knowledge 
as to the initial reaction which leads to the catenary pro- 
cesses culminating in establishing the cleavage-figure with 
which by rhythmical reduplication the development of the 
egg ensues. Hence, the study of the happenings immedi- 
ately ensuing after the mixing of eggs and spermatozoa 



assumes great significance for the understanding of the 
process, fertilization. Moreover, as we shall see, in this 
study inheres another value, since these happenings are of 
prospective significance for the whole range of consecutive 
form-changes by which from the egg the animal emerges, 
changes which taken together we speak of as the egg's 

The evidence summarized in the preceding chap- 
ter makes it clear that in fertilization it is the egg-cyto- 
plasm that reacts with the spermatozoon. A recital 
of this evidence is here unnecessary; one fact noted we 
may recall. This is that whilst no animal sperm-cell 
is capable of fertilizing an egg except as a spermatozoon — ■ 
a sperm-cell which has completed both maturations and 
has become transformed from a spermatid into a spermato- 
zoon by nuclear condensation and remarkable cytoplasmic 
changes — the egg, depending upon the species, has capacity 
for fertilization before, during or after maturation. Thus 
the fertilizability of all animal eggs hangs together with 
some condition in the cytoplasm of the egg and is independ- 
ent of its nuclear state, as germinal vesicle, as first or second 
maturation-nucleus or as a completely matured nucleus. 
This fact would still stand were the events in the ensuing 
fertilization-process the same in all eggs; it becomes of 
paramount significance since we can not reduce these events 
to a common underlying principle which holds for all 

Some change, then, supervenes in the egg-cytoplasm 
which transforms it from a condition of non-fertilizability 
into one of fertilizability. Since we intend to examine the 
reaction between egg and spermatozoon at the moment of 
insemination, and since we know that spermatozoa are at 
this time alike with respect to fertilizing capacity, our 
task concerns itself with the fertilizable condition of the 

is 5 


The fertilizable condition in animal eggs arises suddenly. 
Delage 1 demonstrated that with break-down of the germinal 
vesicle the egg of a starfish undergoes a change which ren- 
ders it fertilizable, a finding which I have confirmed in the 
same and in two other species of the same genus, Asterias. 
Further, not only are these eggs unfertilizable while the 
germinal vesicle is intact; attachment and entrance of the 
spermatozoa during this stage actually inhibit completely 
the break-down of the germinal vesicle. Other eggs which 
normally are extruded into the sea-water in the stage of 
intact germinal vesicle are fertilizable only after its disrup- 
tion, an event which occupies a few minutes. 

The fertilizable condition can not, however, in all eggs 
be correlated with break-down of the germinal vesicle, since, 
as we have seen, eggs like that of Nereis^ for example, and 
of many other animals, are fertilizable only in the germinal 
vesicle stage. The egg of Nereis offers another example of 
the sudden onset of fertilizability. This is shown by the 
following: Eggs of this worm are .normally shed when the 
animals are in the so-called heteronereis phase, i.e., when 
during a period of full-moon they swim actively at the sur- 
face of the sea, at which time all the eggs are in the optimum 
fertilizable condition. Now I have reared in the laboratory 
to the heteronereis phase many of these worms which had 
been collected while immature (i.e., in the nereid phase). 
On five successive days before full-moon I have inseminated 
eggs removed from these worms without obtaining develop- 
ment despite the fact that under the microscope the eggs 
resembled fertilizable ova. 2 On the day of full-moon, eggs 
taken from the same animal, from which others had been 
removed during previous days and inseminated without 

1 Delage, 1901a. 

2 // a fully ripe female {one in the swimming stage) be punctured 
all of her eggs are usually extruded. After the same degree of 
puncture of an immature female only few eggs exude from the site of 



success, fertilized normally and gave normal development. 
Comparable results were obtained with eggs of Platy nereis; 
never did I observe copulation of the animals and the con- 
sequent laying of fertilized eggs before full-moon. 

Often during the early days of its breeding season, the 
sea-urchin, Arbacia, contains eggs with polar bodies 
attached, a condition which indicates that maturation was 
only then ending; usually, normally shed eggs show no polar 
bodies. Such eggs with polar bodies either are not fertiliz- 
able at all or only in small numbers. During one season 
I followed the development of the ovaries of this sea-urchin 
daily beginning in April through to the first of-July. Only 
after the eggs had passed the maturation-stages which 
occupy a brief period, are they fertilizable. 

The fertilizable condition hangs upon or comes with a 
change which occupies a mere point of time in the egg's 
history: before it the egg is unfertilizable and after, fertiliz- 
able. This is doubtless true also for eggs like Otomesostoma, 
DinophiluSj Saccocimts. The spermatozoa enter these 
eggs when they are very young ovocytes, i.e., before the 
eggs' growth-stage. Only when these eggs are fully grown 
and matured, which state they attain some time after the 
precocious sperm-entry, can these eggs be said to be fer- 
tilized. Presumably here also something happens in the 
egg so that it passes from the condition of unfertilizability 
to that of fertilizability. 

The fertilizable condition may endure for hours or even 
for three days as in eggs of sea-urchins, depending upon 
the species and for any given species upon the temperature; 
it persists longer at lower than at higher temperatures. In 
eggs of the flat sea-urchin, Echinarachnius, its duration is 
very short compared with that of Arbacia at the same 
temperature. 1 Eggs of other animals, fertilized outside 

1 By frequently changing the sea-water of the same temperature 
in which the eggs lie, the duration of the fertilizable condition becomes 

iS 7 


of the female's body, as those of echinoderms other than 
sea-urchins, of worms, of molluscs, of ascidians and of 
vertebrates may retain their fertilization-capacity for 

With one exception species of animal eggs undergo no 
change in maturation whilst the fertilizable condition per- 
sists but remain unless fertilized until death in that stage 
in which fertilization normally occurs. Eggs of the genus, 
Asterias, (starfish) make the exception, as pointed out. 
When normally laid by the females, these eggs are in the 
stage of the breaking-down germinal vesicle, their normal 
stage for fertilization. The steadily accumulating evidence 
to show that in normal conditions for breeding the males 
and females of starfishes closely congregate prior to the 
shedding of spermatozoa and eggs also indicates that the 
optimum stage for fertilization is that found in the eggs 
when shed. But if shed eggs or those removed from an 
ovary with their germinal vesicles intact are brought into 
normal sea-water without spermatozoa, they may undergo 
complete maturation. Such eggs fertilized during stages 
of first maturation develop normally; thereafter, fertiliza- 
tion-capacity steadily falls off. After complete maturation 
only low percentages both of fertilization and of subsequent 
development are obtained and are highly abnormal. 

The Abbe Spallanzani made interesting observations on 
the duration of the fertilizability of frog's eggs after deposi- 
tion. Sobotta points out that fertilization of the eggs of 
Amphioxvs succeeds best when the eggs exude from the 
female into sea-water containing spermatozoa; whereas the 
addition of spermatozoa to eggs already in sea-water tends 
to result in abnormal fertilization — an observation indi- 
cating the short duration of fertilizability in this egg. 
Fertilization of the eggs of a fish, the wall-eyed pike, drops 
from 40 per cent, for the eggs that have lain in water for 
two minutes to zero per cent, for eggs that have been in 



water for ten minutes. 1 Where copulation takes place 
between male and female before egg-laying and insemination 
outside of the female's body, the fertilizable condition may 
pass off rapidly. Thus, in eggs of the minnow, Fundulus 
heteroclitus, a bony fish, the fertilizable condition is at its 
height as the eggs extrude during the time, when as the male 
clasps the female, spermatozoa are shed. If one removes 
the female during the act of copulation, then gently presses 
her in order to obtain eggs and places these in sea-water, 
one finds that with residence in sea-water the eggs' capacity 
for fertilization diminishes. The normally shed and insemi- 
nated eggs show one hundred per cent, fertilization. The 
most interesting case illustrative of a brief duration of ferti- 
lizability is that of the eggs of Platynereis, fertilized within 
the female's body. 

The male and female of this marine worm go through a 
most peculiar type of copulation. 2 The sexually mature 
animals swim at the surface of the sea at night during the 
period from full to new moon of the breeding season (sum- 
mer months) and are easily captured. In the laboratory, 
by bringing a male and a female together in a vessel of sea- 
water, one can observe more closely the normal behavior 
displayed at the surface of the open sea. The rapidly 
swimming male entwines the less active and larger female 
and thrusts his tail into her jaws. She thus takes up the 
spermatozoa which pass from buccal cavity to pharynx and 
through lesions in the wall of the pharynx into the coelom 
where they become attached to the eggs. The eggs are 
immediately laid. In one set of observations on 87 females, 
I found, with the aid of fellow workers who recorded the 
time, that the whole process, from the moment that the 
male entwined the female to the beginning of egg-laying, 

1 Reighard, iSqj. 

2 J ust , 19 1 4. 



consumed only about five seconds. And yet every egg 
laid showed an attached spermatozoon and only one. 
These observations were made soon after capture of the 
worms: even so, the delicate males are apt to suffer some- 
what in handling. In nature, therefore, this period is 
presumably not longer than five seconds and may very 
well be shorter. 

Only a very brief fraction of this period of five seconds 
can be concerned with insemination per se. Most of the 
time is consumed by the act of copulation, by the movement 
of the spermatozoa to reach the eggs, and by the wave- 
like muscular contraction of the female by which the eggs 
are laid. That the fertilizable condition is here of extremely 
short duration is further proved by the fact that eggs can 
not be removed from the virgin female to a volume of sea- 
water greater than that of the mass of eggs and the sea-water 
in turn removed quickly enough to insure fertilization. In 
other words, since it can be shown that sea-water does not 
impair the fertilization-capacity of the spermatozoa, it 
must impair very quickly that of the eggs. But the instant 
that the spermatozoon becomes affixed to the egg, the sea- 
water is harmless. 1 

The cytoplasm of eggs, then, at one or another stage of 
maturation becomes suddenly fertilizable and remains so 
for a longer or shorter period of time. Eggs die unless 
fertilized, without exhibiting any change in nuclear state. 
With fertilization, they pass beyond the fertilizable condi- 
tion. I know of only one report, 2 based on insufficient 
evidence, which claims that fertilized eggs can be re-fer- 
tilized. In my experience, eggs having been fertilized lose 
capacity for fertilization for neither they nor their frag- 
ments can be fertilized again. 3 

1 Just, 1915b, 1915c. 

2 Morgan, 1S95. 
* Just, 1923a. 



Fertilizability being resident in the cytoplasm, we may 
ask whether or not it has definite location. As we have 
seen, in the egg of Platynereis, for example, loss of fertiliz- 
ability may occur very quickly. Since an egg is either 
fertilizable or not, it is easy to assume that for eggs generally 
the loss, like the onset, of fertilizability is the event of a 
moment. Now the rapidity of the loss strongly indicates 
the superficial location of that upon which fertilizability 

Two cases show that with the onset of fertilizability the 
ectoplasm of the egg exhibits visible alteration in structure. 
At Naples I often noted that eggs of sea-urchins after com- 
plete maturation but before they are fertilizable show very 
minute projections from their surfaces. Fertilizable eggs, 
in contrast, present a smooth contour; the ectoplasm is 
homogeneous in appearance. The egg of Chaetopterus 
undergoes a series of remarkable changes, its ectoplasm 
flowing from the animal pole to cover the hitherto exposed 
vegetal pole, before it becomes fertilizable by spermatozoa 
entering at the vegetal pole. With the flowing movements 
in the ectoplasm, fertilization may take place at other 

No one has to my knowledge made a systematic study of 
a great number of species of eggs with respect to the occur- 
rence of structural changes in the ectoplasm which might 
be correlated with fertilizability. Until this has been done, 
we can not reject the possiblity that such changes generally 
intervene. Also, these changes may be of such extreme 
delicacy that they are overlooked. Finally, the change to 
the fertilizable condition may not always express itself as 
visibly structural. 

The rapidity with which fertilizability arises and disap- 
pears points to the conclusion that it is a condition of the 
ectoplasm. Then, also, we may assume that fertilization 
is concerned with the ectoplasm. But we need not rest 



content with mere assumption; evidence at hand amounts 
to proof that the chief event in fertilization is a reaction 
between the egg's ectoplasm and the spermatozoon. This 
evidence will now be set forth. 

The proposition that the main event in fertilization is a 
reaction between egg-ectoplasm and spermatozoon is 
supported by the following: (i) The ectoplasm is necessary 
for fertilization. (2) The onset and loss of fertilizability is 
correlated with the appearance and disappearance of an 
ectopias mic substance. (3) Specificity in fertilization 
depends upon the integrity of the ectoplasmic layer. 
(4) Polyspermy obtains when the ectoplasm is slow in 

I first cite an observation of my own. 1 

If uninseminated eggs of the flat sea-urchin, Echinarach- 
nius, stand in shallow dishes of sea-water, they undergo 
a change due to the increasing salinity of the sea-water 
caused by evaporation, a change which manifests itself by 
an alteration of the eggs' surface. This same change is 
induced by placing the eggs in sea-water made hypertonic 
by the addition of sodium chloride (6 parts of 2}i M NaCl 
plus 50 parts sea-water). In either case the surface-layer 
of the eggs on return to normal sea-water is seen to be of a 
thickened and translucent jelly-like nature. Many of these 
eggs, having thus been exposed to hypertonic sea-water, 
under pressure, as by forceful ejection from a pipette, form 
each a protrusion. These vary in size. By other methods 
eggs may be induced to form protrusions; often they show 
them after having lain in normal sea-water for some time. 
Eggs of other sea-urchins, Arbacia, Strongylocentrotus y 
Echinus, Echinocardiam, treated with hypertonic sea-water 
likewise form these protrusions when they are brought into 
normal sea-water. The explanation of the formations is 

1 Just, 1923a- 



this: they represent extruded endoplasm escaping through 
the ruptured surface-layer of the egg, and occur when this 
layer has been so altered that it loses the elasticity so char- 
acteristic of it on the normal unfertilized egg. The resi- 
dence in concentrated sea-water brings about abstraction of 
water from the eggs as revealed by their shrinkage and the 
closer apposition of their cytoplasmic inclusions. The 
surface-layer also loses water; its greater visibility is owing 
to its altered structure: on the normal unfertilized egg it 
appears homogeneous, on the treated egg it appears as a 
system of fine cytoplasmic prolongations attached to the 
vitelline membrane. When these treated eggs are brought 
suddenly into normal sea-water, this altered ectoplasmic 
surface ruptures; and the endoplasm flows out as a cohering 
bud. This outflow is checked as the eggs come into equi- 
librium with their normal medium. 

Every egg with a protrusion, then, is made up of two com- 
ponents, one possessed of the altered surface-layer, the 
other, an endoplasmic bud, devoid of it. Following insemi- 
nation, that portion of every egg enclosed by ectoplasm 
separates a membrane, goes through cleavage and develops 
into a swimming larva. The endoplasmic bud undergoes 
no change; it can not be fertilized. As an egg with a bud 
of endoplasm develops, the bud persists as a mass of undif- 
ferentiated intact material which finally disintegrates. 

The size of protruded endoplasm is without significance; 
it may be extremely minute, of the size of a polar body, 
or it may contain most of the egg-substance. In the latter 
case only the minute component with ectoplasm separates 
the membrane and cleaves, the cleavage resembling the 
so-called disocidal cleavage which is normal for the eggs of 
many animals; but such eggs never form the typical inver- 
sion, invagination, of cells by which the egg becomes a cup- 
like swimming form, composed of two cell-layers. It is 
interesting to note that this invagination (a form of gas- 



trulation) when it occurs — in eggs with buds equal to or 
smaller than the egg-component having ectoplasm — takes 
place at the pole where the bud is formed. This fact might 
mean that the endoplasm is always extruded from the area 
in the uninseminated egg which is destined to be the site of 
invagination. But it is also probable that the endoplasm 
extrudes from any region of the egg, which means that the 
site of invagination becomes prc-determined by extrusion 
of the endoplasm. Whilst sea-urchins' eggs lend them- 
selves beautifully to this mode of endoplasmic extrusion, 
other eggs do not because of the nature of their surfaces 
and the changes taking place in them after experimental 
treatment. In these the vitelline membranes must be 
punctured before the endoplasm can protrude. Such 
injury leads at once to break-down of the egg-cytoplasm, 
as happens when one cuts into the tough membrane 
enclosing the egg of Nereis. 

In the egg of the starfish one can demonstrate that the 
egg-surface plays in fertilization a role similar to that of 
the surface of sea-urchins' eggs. Observations reported 
by Whitaker 1 make it clear that in fertilization of the star- 
fish egg, the effect of the spermatozoon is conducted only 
by the egg-surface. 

The conclusion reached from these observations, that the 
ectoplasm is necessary for fertilization, though it is defi- 
nitely proved for only a few eggs, may nevertheless hold 
for eggs generally, since in them all some kind of surface- 
change follows sperm-attachment. We* should not, to be 
sure, make a virtue of the necessity imposed upon the 
spermatozoon that to effect fertilization it must first make 
contact with the egg-surface; we must know that this par- 
ticular surface is something peculiar because of special 
endowments which set it apart from the endoplasm. 

Whitaker, igjf. 



Mere location does not make the ectoplasm and its behavior 
peculiar, nor the fact that in all cells cytoplasm becomes 
converted into ectoplasm. If the ectoplasm plays this 
role in fertilization, for egg-cells this conversion of cyto- 
plasm into ectoplasm must take place in such wise that 
new and stimuli-receiving substance comes to the surface 
of unfertilized eggs as they pass from the unfertilizable to 
the fertilizable condition. We possess some evidence for 
the appearance of such a substance in the surface-layer 
of the egg with the moment that it becomes fertilizable. 

According to the fertilizin-theory of Lillie, eggs (of sea- 
urchins and of Nereis) in the fertilizable condition contain 
a substance, fertilizin, located at their surfaces which is 
necessary for fertilization. Thus, the statement, no fertili- 
zation without ectoplasm can be amended to read in these 
cases, no fertilization without ectoplasm-located fertilizin. 
Whilst the presence of fertilizin has not been demonstrated 
for all eggs, its occurrence is not limited to those of the forms 
studied by Lillie. I have detected its presence in eggs of 
other sea-urchins and of two other worms {Platynereis 
me galops and P. dumerilii). It has been found in the egg of 
Ciona, an ascidian. I have elsewhere reviewed the work of 
fertilizin; 1 to this review together with Lillie's original 
papers the reader is referred. 2 Here I give only a resume 
with special reference to the ectoplasm-located fertilizin. 

Fertilizin is held to be a mid-body in the reaction between 
egg and spermatozoon. Without it, in the eggs named, 
fertilization does not take place. No cells other than eggs 
possess it and these only when fertilizable. Neither in 
immature nor in fertilized eggs can its presence be detected. 
Eggs treated with means that induce the surface-changes 
identical to those induced by spermatozoa likewise do not 

1 Just, 1930c. 

2 Lillie, 1913, 1919. 



contain it. Repeated changing of the sea-water in which 
eggs lie can wash them free from it so that fertilization is 
no longer possible. Also, by bathing the eggs in sea-water 
containing the animals 5 body fluid, the fertilizin present in 
them can be bound: fertilization is inhibited. 

That washing the eggs removes the fertilizin is evidence 
of its ectoplasmic location. Further, I found that after 
eggs in sea-water containing body-fluid have been mixed 
with spermatozoa, the spermatozoa attach themselves to 
the ectoplasm and even penetrate it but do not fertilize the 
eggs. 1 If however these eggs are soon thereafter thor- 
oughly washed, they develop. From these facts it can be 
concluded that the inhibition to fertilization, by the block- 
ing of fertilizin by body-fluid, takes place in the ectoplasm. 
Moreover, the rapid loss of fertilizing capacity following the 
ectoplasmic changes which underlie the separation of the 
vitelline membrane whether induced by spermatozoon or 
by other means, indicates that the fertilizin is located in 
the ectoplasm. 

The presence of fertilizin can be detected only if some of 
it escapes from the egg into the surrounding sea-water. 
The means of detection is simple. Sea-water in which 
fertilizable eggs have been lying has marked effects on sper- 
matozoa of the same species. If a drop of this sea-water 
be added to a dense suspension of spermatozoa, they are 
first stimulated to intense activity and rushing together 
adhere in masses by means of their heads. This agglutina- 
tion endures for some seconds — or minutes, if the sea-water 
is highly charged with the agglutinating substance, fer- 
tilizin — and then passes off. This agglutination-reaction 
is a striking and interesting phenomenon capable of nicely 
quantitative study. Its value is that of an indicator of 
the presence of the fertilizin that escaped from the eggs; 
it may itself have no significance for fertilization although 

1 Just, IQ22C, iQ2jb. 



it may suggest that the initial stage in the reaction between 
egg and spermatozoon is the agglutination of spermatozoon 
to egg-substance. The agglutination here discussed is 
never induced by treating spermatozoa of one species with 
sea-water charged with the spermagglutinin from eggs of 
another. This, hetero-agglutination, offers a different pic- 
ture qualitatively and quantitatively and is due to a sub- 
stance in the body-fluid. 1 Thus fertilizing as indicated by 
the agglutination-reaction which it induces, is specific. 

No question in the whole problem of fertilization has 
more profound significance than this of specificity. Fre- 
quently students of fertilization have asked themselves the 
question: how is it that in the sea, eggs of one species are 
fertilized by specific and not by foreign spermatozoa ? In 
other words, how do eggs maintain fertilization-specificity? 

In the laboratory fertilization by foreign spermatozoa 
of most marine eggs almost never occurs as a normal event. 
If one mixes eggs of a sea-urchin and of a worm and insemi- 
nates them with spermatozoa of either sea-urchin or worm, 
only specific fertilization occurs. Also, a mixture of two 
kinds of spermatozoa added to eggs belonging to only one 
species of them induces species-fertilization. It has gen- 
erally been found that before cross-fertilization takes place 
among sea-urchins, starfishes, etc., the eggs need either a 
previous treatment with alkali or heat, or long residence 
in sea-water, so that they become " stale, " or an insemina- 
tion with an excessive number of spermatozoa. All these 
methods for inducing cross-fertilization can be shown to 
injure the egg-surface, and so to decrease the egg's vitality. 
As long as the integrity of the ectoplasm remains unim- 
paired, cross-fertilization fails. 

Specificity in fertilization closely resembles that in immu- 
nity-reactions, where a specific anti-toxin combines with 
specific toxin. It is far more specific than an enzyme- 

1 Just, 1919b. 



reaction; besides, there is no evidence that the sperma- 
tozoon initiates fertilization by means of an enzyme, 
though enzyme-reactions certainly follow after fertilization. 

As I see it, specificity in fertilization depends in part 
upon the chemical constitution of the blood or body-fluid 
of the species which produces the eggs and spermatozoa. 
Whilst the chemical constitution of the egg of a species 
differs from that of all other cells comprising the animal's 
body, nevertheless there is a chemical similarity among all 
cells of the animal's body including the egg, inasmuch as 
they are all nourished by the same body-fluid. Species- 
spermatozoa have the identical chemical structure as eggs 
to the extent that they are products of the same species' 
body and blood; at the same time they differ chemically in 
other respects from eggs. Blood (or body fluid) is different 
from all cells including eggs and spermatozoa. Now where 
blood inhibits species-fertilization, this inhibition is owing 
to its being present in such amount that it makes inert 
the specific substance in the egg's ectoplasm. A smaller 
amount blocks foreign spermatozoa and this amount can 
not be wholly removed except by injury of the ectoplasm. 
In nature, owing to the breeding habits of animals, eggs 
and spermatozoa of one species tend to be emitted simul- 
taneously within a fairly restricted breeding ground. Thus 
the chances for specific fertilization are enhanced. Where 
copulation takes place, specific fertilization is still more 
highly insured. On the other side, laboratory conditions 
for inducing cross-fertilization could scarcely obtain in the 
open sea. If nevertheless cross-fertilization should happen 
in nature, we can relate it to an injured state of the egg's 

As stated above, spermatozoa do not enter such fertilized 
eggs, or fragments thereof, which are normally mono- 
spermic, i.e., into which only a single spermatozoon enters. 
The prevailing opinion concerning the block to poly-sperm- 



entry has supported the conclusion reached by Fol in his 
study of sperm-entry into the eggs of the starfish and of 
the sea-urchin, that the separation of the vitelline mem- 
brane prevents the entrance of supernumerary spermatozoa. 
The physical and chemical changes which take place in the 
membrane after separation from the egg-surface are cer- 
tainly such as would bar sperm-entry. But if the block 
were purely mechanical, removal of the vitelline membrane 
should make possible the entrance of extra spermatozoa 
into the egg. It has been shown for the eggs of other ani- 
mals, in addition to those of echinoderms, that disruption 
or removal from fertilized eggs of the vitelline membrane 
does not render the eggs any more liable to poly-sperm- 
entry. 1 With membrane-separation the eggs undergo some 
change and it is this change — not its result, membrane- 
separation — which constitutes the block to the entrance of 
additional spermatozoa. Thus this block, which is more 
subtle than the mechanical obstacle interposed by the 
presence of a separated membrane, is established before 
membrane-separation occurs. 

A rather exact indication as to the moment when the 
block intervenes is furnished by my observations on the 
egg of Echinarachnius. Here one can follow the wave in 
the ectoplasm which begins at the point of sperm-entry 
and sweeps over the egg. As this wave progresses, it 
renders the egg immune to the entry of supernumerary 
attached spermatozoa. The behavior of these latter sper- 
matozoa can be observed to change with the entrance of 
the "fertilizing" spermatozoon into the egg: as the wave 

1 By putting inseminated eggs in the best fertilizable condition 
through bolting-silk at the moment when they separate their mem- 
branes one can most easily and with the least harm deprive them of 
their membranes. Spermatozoa may be added immediately or at 
any time thereafter but each egg remains fertilized only by the single 
spermatozoon of the first insemination. 



moves from the site of entry, the movements of those sper- 
matozoa nearest the site slacken and cease; next those 
farther away become immobile; finally those situated 180 
away come to a standstill. These events take place before 
membrane-separation. Often, however, the process is 
much too rapid to allow one easily to follow it. Neverthe- 
less I have under a good apochromatic lens followed it in 
eggs in best condition taken from hundreds of specimens. 

The eggs of Arbacia, in my experience, if in best condition 
are never polyspermic. It is possible to fix eggs one second 
after spermatozoa have been added to them; examined 
under the microscope, each egg shows a spermatozoon 
attached to it. If a thick sperm-suspension be added to 
the eggs as early as one second after the first insemination, 
no polyspermy occurs. I have also made the initial insemi- 
nation with the heaviest sperm-concentration procurable, 
i.e., "dry" sperm as it exudes from the male, and have 
obtained only mono-spermic fertilization. Thus, the block 
to polyspermy is most rapidly interposed. 

The eggs of Platynereis Avhen laid have each one sperma- 
tozoon attached. And yet in the body-cavity of the worm, 
the eggs, especially those in the anterior segments, are 
in the presence of supernumerary spermatozoa, as sections of 
the worms made immediately after copulation reveal. In 
all my slides of normally laid Platynereis eggs I have rarely 
seen one egg on which two spermatozoa are attached. 1 
have never seen more than one spermatozoon within an egg. 

Surely under the conditions of the normal insemination 
of eggs of Platynereis one can not postulate a mechanism so 
precise that it would distribute the spermatozoa in such 
wise that each egg would receive only one spermatozoon. 
Rather, within the narrow closed space of the body cavity 
packed with eggs — even the head segment contains them— 
prevails a situation most favorable for the aggregation of 
supernumerary spermatozoa around each egg. And yet 



polyspermy does not occur. Eggs in the anterior segments 
of the worm, where most spermatozoa are found, are as free 
from polyspermy as those which first issue from the anal 

My preparations of starfish eggs which show more than 
one spermatozoon entering are of eggs fertilized either before 
break-down of the germinal vesicle or after second matura- 
tion. Never in the stage of first maturation when the eggs 
are in the stage of optimum fertilizability is there poly- 
sperm-entry. If, however, fertilizable eggs be injured, 
supernumerary spermatozoa enter. 

These observations lend themselves to the conclusion 
that the block to polyspermy is rapidly established with the 
attachment or entry of one spermatozoon. 

Now into all these eggs — of Echinarachnius, Arbacia, 
Platynereis, Asierias — the spermatozoon may enter at any 
point. They thus differ from some other monospermic eggs, 
"as" those of the trout or the ink-fish, which possess a tube, 
the micropyle, through which the spermatozoon gains 
entrance; or such eggs, as of Ciona and of Amphioxus, into 
which spermatozoa enter always at the vegetal pole. 
Theoretically, therefore, these eggs under discussion are 
polyspermic; until they establish the block against super- 
numerary spermatozoa any part of the eggs' surface is 
responsive to sperm-attachment — leaving aside the unlikely 
assumption that one site alone admits the spermatozoon 
and that this would vary even in eggs of the same female. 
From this point of view the interposition of the block to 
polyspermy becomes highly significant for it offers a means 
by which we can set the termination of the initial reaction 
between normal egg and normal spermatozoon of any of 
the species named: it ends with sperm-attachment. 1 The 

1 This refers to normal eggs, it must be understood, since sperma- 
tozoa may enter unfertilizable eggs — i.e., immature ones. See 
Iwanzoff and others. 



process to all intents and purposes is instantaneous, and 
therefore ectoplasmic. And this reaction that brings about 
a fundamental change in the egg which renders it incapable 
of reacting to other spermatozoa present and initiates the 
egg's development is the fertilization-reaction. 

The rapidity with which the complete fertilization-reac- 
tion terminates, obviously depends upon the speed at which 
the reaction runs. If the egg's ectoplasm is highly reactive 
the reaction will proceed at a rapid rate. In another egg the 
rate normally may be slower and yet sufficiently rapid 
to inhibit polyspermy under natural conditions where eggs 
and spermatozoa are fully normal. In these, experimental 
polyspermy may be induced with greater facility. The 
reaction will be slowest in normally polyspermic eggs. If 
the block to polyspermy also in normally polyspermic eggs 
is taken as the indication that the fertilization-reaction is 
complete, this end would come in such eggs when finally 
they can receive no more spermatozoa. 

Normally monospermic eggs can be rendered polyspermic 
by experimental treatment as staling (long residence in 
sea-water), treatment with alkali, acids or poisons, subjec- 
tion to low temperature, in short, by methods for inducing 
injury or weakness. The eggs also tend to be polyspermic 
when below optimum condition as, for example, those 
obtained toward the end of the breeding season or from 
moribund animals. Polyspermy in them thus is a sign 
of weakness and hence pathological. In all conditions fav- 
orable to poly-sperm-entry the ectoplasmic response differs 
from that in normal fertilization. If, for example, eggs of 
a sea-urchin after having been kept at low temperature, 
around 5°C, are inseminated, they show polyspermy and 
the vitelline membrane separates only very slightly, which 
indicates abnormal ectoplasmic activity. If treated with' 
an organic acid, e.g., 2 cc. Ko normal butyric acid plus 
50 cc. of sea-water, for one minute or more, eggs of sea- 



urchins are rendered highly susceptible to poly-sperm-entry 
whilst their ectoplasm becomes thickened and the mem- 
brane only slightly separated. The same result obtains 
after exposing the eggs to the alkaloids, strychnine, nicotine, 
etc., as the Hertwigs 1 showed in their now classic experi- 
ments. If one repeats their observations one marks the 
striking responses of the egg-surface to insemination, among 
others, the longer duration of the many " fertilization- 
cones/ 5 The ectoplasm need not be severely injured in 
order that polyspermy succeeds. If the ectoplasmic 
response to insemination be slowed down, polyspermy may 
ensue. Thus, if one knows that eggs of a given lot are 
below normal by having learned through trial insemina- 
tions on some of them that the surface-changes underlying 
membrane-separation proceed at an abnormally slow rate, 
one can by inseminating with a heavier sperm-suspension 
than that usually employed secure polyspermy. 

Some animal eggs, as those of cartilaginous fishes, of some 
amphibians and of birds, are normally polyspermic. Poly- 
spermy has also been reported in insect eggs. All normally 
polyspermic eggs are large. 2 But since there exist also 
some large eggs which are not polyspermic, it is, presum- 
ably, not so much the size of the egg but the slow reaction 
of its ectoplasm which makes polyspermy possible. 

The suddenly arising fertilizable condition of animal eggs 
thus resides in the ectoplasm. The indications are that 
this is a chemical reaction. Consider the problem of spe- 

1 Hertwig, 0. and R^ iSSy. 

2 Bonnevie*s conclusion (ipoy) that polyspermy obtains in eggs of 
Membranipora is incorrect as careful study of her paper reveals. 
See also my failure to find polyspermy in this egg — Just, 1934. 
MacBride's statement that polyspermy occurs in eggs of Pedicellina 
is undoubtedly due to his error in interpreting Haischek's statement 
that he often observed numerous motile spermatozoa in the perivitelline 



cificity. The phenomenon of specificity generally we relate 
to chemical constitution and not to physical properties. 
No valid reason can be proffered for assuming that spe- 
cificity in fertilization is unlike that met with in other bio- 
logical processes. Unless such reason is forthcoming, we 
must assume specific fertilization as chemical. Where, as 
in eggs of some species, a substance has been isolated upon 
which fertilization depends, we are warranted again in 
postulating that the fertilization-reaction is chemical — one 
between this substance and the spermatozoon. Finally, 
the changes, so strikingly visible in many eggs, whereby 
the vitelline membrane is separated, can not, as we have 
seen, be regarded as the initial reaction in fertilization: 
membrane-separation, a physical process, though common 
to all eggs, is the result of the fertilization-reaction and not 
the reaction itself. 

What happens is this: the spermatozoon sets off a first 
explosion in the narrow area of the egg-surface which it 
touches, and kindles the spark which leads to a chain of 
explosions. The fertilization-reaction is thus a trigger- 
reaction. A large portion of the surface is shattered by the 
explosive effect with gas-exchange and heat-liberation. 
One can in many eggs see this break-down of the surface 
by which the egg loses substance and the membrane is 
separated. The pressure between egg and membrane, in 
the perivitelline space, probably is considerable as the 
ectoplasmic colloids disintegrate and go into solution. 
Water rushes in and further distends the still ductile mem- 
brane which then sets as a stiff structure. The fully 
separated membrane then becomes brittle; one can more 
easily remove it immediately after its separation than later 
when it becomes tough. But these changes in the mem- 
brane are due to its separation from the egg, for it no longer 
forms part of the living system. What therefore are impor- 
tant for the fertilization-reaction are the underlying surface- 



changes that result in membrane-separation and neither 
the consequent physical act of separation nor the changes 
in the membrane itself. 

Nevertheless this separation of the membrane constitutes 
an easily visible indicator of the underlying surface-changes 
initiated by the fertilization-reaction. It tells us quickly 
whether or not fertilization has taken place. Also, it gives 
us information concerning the quality of the eggs fertilized. 
Membranes that separate incompletely and are not equi- 
distant from the egg at all points and are slow in rate of 
separation mean eggs of poor fertilizability that subse- 
quently develop abnormally. 

Let me emphasize that here I speak only of fertilized eggs 
which have favorable conditions for subsequent develop- 
ment. If after fertilization conditions are deleterious, the 
eggs will not develop normally no matter how perfect the 
membranes. Moreover, as we shall learn in the next chap- 
ter, membrane-separation alone, though most perfect, does 
not guarantee development. Indeed, even in those cases 
in which perfect membranes are separated by experimental 
agents, they are not wholly identical with those called forth 
by sperm. What here looms large is the value of the 
quality of the surface-changes for foretelling the future 
course of the egg's development. Quite apart there- 
fore from their value for the study of the fertilization-reac- 
tion, the ectoplasmic changes by which the membrane 
becomes separated have greatest significance. Develop- 
ment embraces a series of surface-changes which vary as 
the surface-area increases with the march of development. 
Those occurring at the very outset are most striking; they 
mark out the course, direct the way toward the final out- 
come of fertilization, the formation of the complex organism 
out of a single cell, the egg. 


Parth en og en esis 

jDIOLOGICAL processes often reveal themseles as 
themes with many variations. Fugitive incidental nuances 
embellish the process of fertilization, as we have seen; but 
though they run the whole range of variation, they never 
obscure the motive: fertilization as the union of egg-plasma 
and spermatozoon. 

That the egg- and sperm-nucleus are equipotent in the 
developmental process, since with either alone the egg's 
development can successfully proceed, has been pointed out. 
Thus, whilst the development of the egg of any one of sev- 
eral marine invertebrate animals can be initiated by fer- 
tilizing it after its nucleus has been removed, the egg-nucleus 
of the egg of Rhabditis aberrans alone takes part in devel- 
opment, for the sperm-nucleus within the egg remains 
inert. Neither of these examples is a violation of the state- 
ment that for the majority of multicellular animals the life 
of the new individual begins with the coming together of 
the two living gametes, egg and spermatozoon. Rather, 
both demonstrate very clearly this essential fact: fertiliza- 
tion is not the fusion of the egg- and sperm-nuclei but the 
union of egg-plasma and spermatozoon. Now we turn to 
the discussion of phenomena which reveal that even this 
union is not always necessary for the elevation of the life- 
process in the egg from the level of a single cell to that of 
an individual of multicellular organization. There are 
eggs which normally develop without spermatozoa. This 
type of development, encountered among many animal 
species, is called parthenogenesis. Of the theme, the initia- 


tion of the development in the egg, therefore, fertilization 
and parthenogenesis are variations. 

Investigations in the realm of inanimate nature have in 
the last decades led to such a wealth of new discoveries, 
have so shattered the foundations of the old point of view 
that we are warned against beholding the picture of inani- 
mate nature now offered us as final. Full well we know 
that every day holds the possibility of new discoveries 
which must make everything known appear in a new light. 
Also in the investigation of living nature we may be certain 
that we stand only on the threshold of the knowledge of its 
deeper principles and we should avoid too quick and final 

Even when we know clearly the exact goals which living 
nature reaches, and when we survey the chief paths which 
it traverses to reach these goals, always does the fact that 
there are by-paths — detours to our conditioned human 
comprehension — take us by surprise. We should not look 
upon this endless richness of nature's creative capacity as 
an opportunity for us to confirm a preconceived notion or 
theory. Rather with wide open mind should we be recep- 
tive to this richness in all its manifold expressions. 

In calling forth a new individual, nature has not limited 
itself to one mode; both fertilization and parthenogenesis 
initiate development of the multicellular animal. When 
it was found that parthenogenetic development could be 
initiated also in the laboratory by experimental means, we 
should have taken this discovery as a sign of the manifold 
possibilities that inhere in an egg-cell to respond to stimuli. 
Instead, this discovery was held to mean that the final solu- 
tion of the problem of the initiation of development in 
animal eggs had been reached, that the final word concern- 
ing one biological phenomenon had been spoken — and 
even, that man here had mastered nature, that he himself 
could create life. The discovery of isotopes has taught us 



that with respect to even simpler material structure nature 
does not always limit itself to one mode, that we must 
amend our natural laws as we learn more about nature. 
Such discoveries also suggest that more complex natural 
processes wear many guises. Had the parthenogenesis 
that occurs in nature received due attention — if in fact it 
had come within the knowledge of those who so greatly 
busied themselves with experimental parthenogenesis — the 
discovery of experimental parthenogenesis never would 
have had the palm awarded it. 

Parthenogenetic development is found naturally occur- 
ring especially in the groups of rotifers and arthropods. 
This natural parthenogenesis may be fixed (obligatory) — 
i.e., the eggs develop normally only parthenogenetically. 
Experimentally, a change either in the environment or 
of the food of the mother may render the eggs capable of 
receiving the spermatozoon. In such cases the period of 
fertilizability comes during first maturation. Natural 
parthenogenesis is also variable (facultative) — these eggs 
develop normally either parthenogenetically or by fertiliza- 
tion: as, for example, eggs of the honey-bee. 

In fixed parthenogenesis, the first polar body is extruded, 
the second is not; hence the egg develops with the double 
(somatic) number of chromosomes. The chromosomes of 
the second polar body may unite with those of the egg- 
nucleus, thus acting as a substitute for the sperm-nucleus. 1 
An egg of the variable type of parthenogenesis extrudes 
both polar bodies and develops with only a single set of 
chromosomes. A double set of chromosomes is not, there- 
fore, a prerequisite for the onset of naturally occurring 
parthenogenetic development; nor does the presence of 
only a single set of chromosomes imply defective develop- 
ment: the male honey bee (or drone) for instance, partheno- 

1 Cf. Anemia, Brauer, 1S93. 



genetic in origin, is a normal organism though he lacks the 
sperm-borne chromosomes of his sisters. 

With the aid of chemical solutions, changes in tempera- 
ture, mechanical shock or radiations, eggs which normally 
never develop without fertilization can be induced to 
develop parthenogenetically. Up to the present we have 
succeeded in inducing this artificial or experimental par- 
thenogenesis in eggs of echinoderms, worms, molluscs, a 
spider, fishes, and frogs. As we shall later learn, the only 
period in which experimental parthenogenesis is possible 
coincides with the stage in which the egg is fertilizable. 
Since, for the eggs of the animals named, this fertilizable 
period falls, for some of them, in the germinal vesicle stage, 
for others during first, for others during second, and for the 
remainder after complete maturation, we can not regard 
only one of these stages as the prerequisite for induced 

Unfortunately, of the eggs of the various species of ani- 
mals that have been treated with means for inducing par- 
thenogenesis only those of a sea-urchin, of a starfish and 
of frogs have been reared to sexual maturity. The 
composition of the nucleus can generally be given only for 
early stages of development. With respect to the end- 
result of development our knowledge of induced partheno- 
genesis falls far short of that of the natural. Of the few 
cases known the sea-urchins' and the starfish eggs show 
throughout development a single set of chromosomes. The 
adult frog derived from a parthenogenetic egg is different; 
such an individual has either cells with single or such with 
double sets of chromosomes or cells of both types. Noth- 
ing indicates that the normality of these adults varies with 
chromosome-garniture. 1 The same can be said for the 

1 We must not overlook the fact that there are variations in the 
chromosome number in cells of adults from fertilized eggs. 



larval stage of those eggs whose induced parthenogenetic 
development has not been followed farther. 

After successful treatment with a parthenogenetic means 
eggs whose optimum period for fertilization follows com- 
plete maturation develop parthenogenetically with one set 
of chromosomes (example: echinids). The egg of the frog 
after puncture (the method used for inducing partheno- 
genesis in this egg) made after extrusion of the first polar 
body, which is the fertilizable stage for this egg, extrudes 
the second polar body and begins development always with 
only a single garniture of chromosomes, while in later devel- 
opment this chromosome-number has been found doubled. 
The situation is different with eggs physiologically ripe for 
fertilization in either the stage of the intact germinal vesicle 
or that of first maturation. After successful treatment 
with a means of experimental parthenogenesis, they develop 
with or without one or both polar bodies — hence, their 
blastomeres contain single, double or more garnitures of 

It is thus clear that development induced by experimental 
means does not depend upon the presence of two or more 
sets of chromosomes. In this respect experimental par- 
thenogenesis resembles the normal. An explanation of 
either type of development can not, therefore, be based 
upon a bipartite make-up of the first cleavage-nucleus. 
Here we note, however, the following difference between 
the natural and the experimental process: in natural par- 
thenogenesis the egg always has the nuclei of both polar 
bodies available although it develops with or without the 
union of the second of these with the definite egg-nucleus. 
In experimental parthenogenesis the polar-body nuclei are 
not always available for fusion with the egg-nucleus. This 
difference is due to the fact that in eggs which possess the 
capacity for natural parthenogenesis, development, either 
with or without spermatozoon, begins in the stage of first 
maturation, i.e., before polar body extrusion — whilst eggs 



capable of developmental response to experimental means 
are distributed among all of the four categories made above 
with respect to the stage in the maturation process, in which 
animal eggs are physiologically ripe for fertilization. 

Eggs which are fertilizable in the stage of first matura- 
tion are more widespread in occurrence among animals 
than those of any other class. Since both the facultative 
and the fixed parthenogenetic eggs belong to this large 
class, we might expect that experimental parthenogenesis 
is more easily elicited in this class of eggs than in any other. 
This expectation, however, is not fulfilled. Although the 
egg of the starfish, fertilizable in the stage of first matura- 
tion, and the first in which was discovered the capacity of 
eggs to respond to experimental treatment with develop- 
ment, is of all animal eggs the most readily responsive to 
experimental means for parthenogenesis, other eggs of the 
same class, as we shall learn, may fail wholly to respond 
to means effective upon the starfish egg or they respond 
with extremely abnormal development. Also, the greatest 
number of positive results has been obtained on completely 
matured eggs, i.e., those of sea-urchins. Finally, eggs of 
the two remaining classes when properly treated develop 
normally; frogs' eggs, for example, reach the adult stage. 
We can not therefore correlate the ease with which experi- 
mental treatment elicits parthenogenesis with the nuclear 
state of the egg. Whether or not the restriction of natural 
parthenogenesis to eggs fertilizable in the stage of first 
maturation bears a causal relation to this mitotic phase of 
the nucleus, has not yet been determined. As to the cause 
of experimental parthenogenesis, however, there are only 
two possibilities: it rests either in the egg's cytoplasm or 
in the means that brings about the parthenogenetic 

The latter view one often meets, namely, that the means 
carries into the egg something which initiates its develop- 
ment. The brief review of the response of individual eggs 



to experimental means for eliciting development which now 
follows will give evidence against this view and prove that 
the egg's response like that in fertilization is cytoplasmic. 

Above I called attention to the interesting case of the 
starfish egg. Like that of many other eggs, its germinal 
vesicle breaks down when the egg comes from the female 
into sea-water. But whereas other eggs of this class do not 
develop farther unless they are fertilized, the unfertilized 
egg of the starfish completes both maturation-divisions. 
This fact suggests that this egg is extremely unstable and 
lies close to being normally parthenogenetic. It is not 
astonishing, therefore, that the starfish egg is found to be 
most easily induced to parthenogenetic development by 
one of several means. 

The knowledge that sea-water charged with carbon-diox- 
ide brings about the development of eggs of a starfish, 
Asterias glacialis, we owe to Delage 1 who was able to rear 
the parthenogenetic larvae through metamorphosis. This 
method has been successfully employed by others 2 on eggs 
of this as well as on those of other species of Asterias. 
Development also results if one crowds a large number of 
eggs in the stage of first maturation in a small volume of 
sea-water and leaves them there for about an hour — in this 
case the result is due to carbon-dioxide produced by the 
eggs. In my judgment the parthenogenetic development 
obtained by Mathews 3 through shaking eggs of Asterias 
forbesii is likewise to be attributed to carbon-dioxide pro- 
duced by crowding the eggs; shaking of maturing eggs, in 
my experience at least, is without avail unless the eggs be 

If a thin suspension of maturing unfertilized eggs of this 
species is placed in large volumes of sea-water in uncovered 

1 Delage, 190S, igio. 

2 Buchner, igil and others. 

3 Mathews, iqoi. 



shallow dishes so that evaporation takes place, the eggs 
develop when transferred to normal sea-water. Similarly, 
sea-water to which solutions of sodium or potassium chloride 
have been added induces development. Thus, hypertonic 
sea-water is a means of calling forth parthenogenesis. 

Butyric acid in sea-water, like carbon-dioxide, also 
induces parthenogenesis in the eggs of Asterias forbesii. 
R. S. Lillie has studied the relation of the duration of the 
treatment with this acid in sea-water to the degree of 
response elicited. He obtained only membrane-separation 
with short exposure; membrane-separation and cleavage 
with a longer exposure, and full development to the swim- 
ming stage by a still farther increased length of exposure. 
Increased temperature of the sea-water alone or combined 
with butyric acid, and two short exposures to the acid sepa- 
rated by the residence of the eggs in normal sea-water are 
also successful. Any one of these methods is superior to 
the hypertonic sea-water method by giving higher percent- 
age of parthenogenetic development. 

In 1876 Richard Greef reported that he had observed the 
parthenogenetic development of the eggs of a starfish, 
Asteracanthion {Asterias) to the ciliated larval stage (gas- 
trula). The larvae obtained were vigorous and corre- 
sponded thoroughly with those developed from fertilized 
eggs. Since Greef had taken every precaution against the 
accidental presence of spermatozoa, since further the ani- 
mals from which the eggs came gave no evidence of being 
hermaphroditic, 1 and since, as he had learned before, ferti- 
lized eggs reach first cleavage in one to two hours after 
insemination, whilst these parthenogenetic ones cleaved 
first only at ten to twelve hours after having come into sea- 
water, he was certain that he had observed a true case of 

1 Hermaphroditism among starfish is rare. See Retzius, igi /, 
Vol. 16 of his collected works; Buchner^ ipli. 



parthenogenesis. That he had induced parthenogenesis 
and had not followed a normally parthenogenetic develop- 
ment seems to me to be beyond question. Thus without 
knowing it he was the first to induce parthenogenesis in 
marine eggs. Evidence may be adduced upon which this 
judgment is based. 

In the first line I place Greef's work itself. The observed 
great difference between the fertilized and the partheno- 
genetic eggs with respect to the time when they reach first 
cleavage, points strongly to an experimental induction of 
development. This was probably brought about by an 
altered condition of the sea-water. If the eggs were 

crowded, carbon-dioxide was pres- 
ent in high concentration. At 
the time of year, the beginning of 
May, when the observations were 
made, the water in the vessels 
containing the eggs most probably 
rose in temperature, in the absence 
of any precautions taken against 
such rise. Or, if the eggs were 
kept in uncovered vessels, evapor- 
ation took place. Any one of 

Fig ~\i — Ripe egg of Aster- 
acanthion rubens after having these possibilities, all Well-knOWll 

lain two hours in sea-water means for inducing parthenogenesis 

(after Schneider). * r i i j 

v J in starfish eggs, would account 

for Greefs results. Schneider in 1883 pictured an egg of 
Aster acanthion which after having lain two hours in sea- 
water showed many asters. This is clear evidence to any- 
one familiar with this egg that this change is induced by 
the residence of the egg in sea-water (Fig. 32). 

Secondly, later work on this egg may be considered. In 
reading 0- Hertwig's report on his observations made four- 
teen years after Greefs, on the egg of Asterias glacialis and 



especially on that of Astropecten^ one is struck more by his 
failures than his success to obtain parthenogenesis. 1 Were 
the eggs of these two starfishes normally parthenogenetic, 
he would have secured a higher per cent, of development; 
also, the development would have gone farther and would 
have more closely resembled that of fertilized eggs. Three 
years earlier, when he attempted to observe parthenogenesis 
in the eggs of Asterias and so to repeat Greef s work, he 
had either changed or aerated the sea-water, so that the 
sea-water neither increased in hypertonicity nor in tempera- 
ture and did not become charged with carbon-dioxide. 
At that time he failed completely to secure development 
beyond the establishment of the definite egg-nucleus. In 
other words, the eggs, as is normal for them, merely com- 
pleted maturation in sea-water. In the later experiments 
where he observed parthenogenetic development, he does 
not mention having protected the eggs against changes in 
the sea-water. The one egg that reached the blastula- 
stage, though seemingly normal in outward appearance, 
lacked the separated membrane so characteristic of the 
fertilized egg and of that which has been induced to develop 
by means of treatment with C0 2 or with increased tempera- 
ture. This observation on the single blastula obtained, 
strongly indicates in the light of my experience that Hert- 
wig induced parthenogenesis by means of sea-water made 
hypertonic through evaporation. 

The strongest evidence that both Greef and Hertwig 
induced parthenogenesis experimentally in these eggs lies 
in the fact that although starfish eggs are extremely 
responsive to experimental means, no one since has suc- 
ceeded in demonstrating that they are normally partheno- 
genetic. My experience with the egg of Astropecten 

1 Hertzoig^ 0., iSqo. 



convinces me that it resembles those of three species of 
Asterias which I have studied, and that its classification as 
a normally parthenogenetic egg is unwarranted. 

Eggs of the marine worm, Chaetopterus, like those of the 
starfish fertilizable in the stage of first maturation, when 
treated with hypertonic sea-water develop into bizarre and 
wholly abnormal swimming forms through repeated nuclear 
divisions in the cytoplasm which fails to divide, a condition 
known as differentiation without cleavage. 1 Following 
exposure to hypertonic sea-water, other eggs fertilizable 
in the stage of first maturation, e.g., those of Podarke 2 and 
of Amphitrite* both worms, develop without cytoplasmic 
cleavage. For the egg of Chaetopterus and for that of the 
small marine clam, Cumin gia, increased temperature of 
the sea-water, induces the best type of parthenogenetic 
development — with cytoplasmic as well as nuclear divisions. 

The egg of Chaetopterus merits attention because Mead 
used it in his interesting experiments. Previously in 
his paper on the normal fertilization-process in this egg 
he had set forth a strong argument against the theory 
that fertilization is due to the introduction of centrosomes 
into the egg by the spermatozoon. His work on the effect 
of salt solutions in initiating development was the logical 
outcome of his main conclusion in the work on fertilization, 
a later statement of which reveals his clear conception of the 
initiation of development as a chemical process. His work 
on inducing parthenogenesis, therefore, was no mere acci- 
dental experiment; rather, at its basis was a well defined 
working hypothesis. His was the first work based on the 
assumption that development can be experimentally 
induced. Why Mead so suddenly dropped this promising 
line of research is strange. It was justly said by Loeb, 

1 Lillie, 1906. 

2 Treadwell) 1902. 

3 Scott, 1906. 



that Mead's work is too often overlooked and deserves 
greater appreciation than it has received. 

Turning to that class of eggs which are fertilizable in the 
stage of the intact germinal vesicle, I may cite the results 
of experiments to induce parthenogenesis in three eggs: 
those of Mactra, of Thalassemia and of Nereis. 

Eggs of Mactra, a marine clam, exposed to 10 cc. of 232 
N KC1 plus 90 cc. of sea-water for 45 minutes complete 
maturation and develop as far as the six-cell stage. Longer 
exposures, ij 2 to 4 hours, and to stronger solutions, give 
development without formation of polar bodies. KC1- 
treated eggs may also develop into swimming forms by 
nuclear divisions onlv, i.e.. thev differentiate without cleav- 
age as do the eggs of worms mentioned above that are 
similarly treated. 1 

By the action of either mineral or organic acids, including 
carbon-dioxide, in sea-water, one can induce the develop- 
ment of 50 to 60 per cent, of unfertilized eggs of Thalas- 
sema mellita* a marine worm, to the larval stage; this is 
scarcely to be distinguished from that of the normal, i.e., 
developed from fertilized eggs. Because of these positive 
results obtained with acids, the effects of hypertonic sea- 
water were not thoroughly investigated. Those reported 
are not only too incomplete but also too contradictory to 
permit the drawing of a conclusion. In view of the effi- 
ciency of hypertonic sea-water on so many other eggs, how- 
ever, one may hazard the opinion that it is efficacious on 
this egg also. One is strengthened in this opinion by recent 
work on the eggs of another species of Thalassemia, T. 
neptuni. z 

In the egg of Nereis by shaking or centrifugal force ecto- 
plasmic break-down, with extrusion of jelly, and matura- 

1 Kostanecki, 191 1 and earlier. 

2 Lefevre, 1901 . 

3 Hobson, 192S. 



tion are brought about- Prolonged exposure to weak or 
short exposure to stronger hypertonic sea-water gives the 
same results. With still longer exposure to hypertonic sea- 
water, differentiation without cleavage is called forth. By 
treatment with hypertonic sea-water I have never been 
able to induce development with cytoplasmic cleavage in 
this egg. Hypotonic sea-water also gives differentiation 
without cleavage. 1 I find that the percentage of swimming 
forms may be increased by the addition of an acid or an 
alkali to the dilute sea-water. Hypotonic sea-water in my 
experience is an inferior means of inducing parthenogenesis 
in this egg. Ultra-violet radiation also induces a qualita- 
tively poor development. 2 If eggs from a female from 
whose body the sea-water has been removed, are placed on 
a dry glass plate for a few moments, and are then put in 
normal sea-water, a small percentage develops. 3 Eggs 
having been kept at a temperature around 5°C. extrude 
jelly at once when removed directly to normal sea-water at 
room temperature (around 20°C). Of these some will 
develop farther. The best method for inducing the devel- 
opment to the stage of larval worms, which can scarcely 
be distinguished from those developed from normal, fer- 
tilized eggs, is to expose eggs to sea-water at a temperature 
of from 30 to 33°C. 4 In this method it is necessary that 
the eggs come directly from the female into the warm 
sea-water without having lain in sea-water at room tempera- 
ture. If eggs are taken from sea-water at room tempera- 
ture, they need an exposure to a higher temperature, around 
40°C, for about a minute to be stimulated to develop. 5 In 

1 Just, IQ2Se. 

2 Just, I933C. 

3 Just, 1915a. 

4 Ibid. 

b Just, unpublished observations. 



this case the percentage of development is not so high as 
that obtained by use of the method just mentioned. 

Eggs of sea-urchins, fertilizable after complete matura- 
tion, early became the favorite objects for experiments on 
induced parthenogenesis. 

Although Morgan made experiments which indicated that 
by means of treatment with hypertonic sea-water, eggs of 
Arbacia can be induced to develop parthenogenetically, he 
failed to extend his studies sufficiently and thus to obtain 
the production of larval forms from unfertilized eggs. 
Despite this failure his work demonstrated that treatment 
with hypertonic sea-water can call forth the establishment 
of the mitotic complex, the sign that development has been 
initiated. By extending Morgan's work, J. Loeb was able 
to induce eggs of Arbacia to develop parthenogenetically to 
swimming forms. 1 He deserves the full credit because he 
appreciated his findings, whilst it appears that of his pre- 
decessors, Greef, O. and R. Hertwig, Adead and Morgan, 
only Mead experimented with the definite purpose of induc- 
ing development by chemical means. 

Loeb found that, whilst after exposure for two hours to a 
solution of 50 cc. of sea-water plus 50 cc. 1 % M MgCK, 
the eggs of the common sea-urchin, Arbacia, found at Woods 
Hole, Mass., developed into swimming larvae, the use of 
other salt solutions gave no results. The next year, work- 
ing on the California coast with eggs of the sea-urchins, 
Strongylocentrotus purpuratus and S. franciscanus, he was 
able to induce development not only with MgCU, but also 
with NaCl or KC1, either of these salts being added to 
sea-water, in the proportions 10 cc. of a 2.5 gram molecular 
solution to 90 cc. of sea-water. Later, other salts as well as 
cane sugar and urea were also found to be effective. This is 
the so-called old or original method of parthenogenesis, 

1 See Loeb 1913 and earlier. 



sometimes called the osmotic method for inducing develop- 
ment of the unfertilized sea-urchin egg. 

This early work established that sea-water, if made suffi- 
ciently hypertonic by the addition of electrolytes or non- 
electrolytes, is capable of initiating development of 
sea-urchin eggs. It shows that the effective agent is not 
specific; the original failure to induce development with 
the chlorides of sodium and potassium was obviously due 
to an error which Loeb made in preparing the solutions. 
It was soon learned that sea-water concentrated by boiling 
is capable of inducing the development of this egg. I have 
been able to induce experimental parthenogenesis simply 
by allowing the eggs to remain uncovered in a glass dish 
with sea-water; in this way, sufficient evaporation takes 
place to bring the sea-water to that degree of hypertonicity 
which is effective for stimulating the eggs to develop. 1 

But there are certain shortcomings to this osmotic 
method. As we have seen, the eggs of sea-urchins separate 
their vitelline membranes after insemination. The final 
distance of the membrane from the egg and the rapidity of 
this separation are indices of the physiological state of the 
egg: an egg in best condition separates its membrane at a 
uniform and rapid rate, with the result that it is equidistant 
from the egg-surface at all points. 2 After the above men- 
tioned treatment with hypertonic sea-water, sea-urchin 
eggs do not show a separated membrane. Instead, the 
vitelline membrane present on the egg before treatment 
remains closely stuck; the hyaline plasma-layer beneath it 
swells. 3 Furthermore, the fertilized egg, if it be in opti- 
mum condition, cleaves at a regular tempo and the blasto- 
meres adhere to each other. After the treatment with 
the hypertonic sea-water here discussed the blastomeres 

1 Just, 1928a. 

2 Just, 1928c. 

3 Just, 1919c, 1922b. 



cleave irregularly, both as to tempo and as to size, and they 
tend to fall apart. In the next place, normally the fertilized 
egg develops into a form which swims at the surface of the 
sea-water; subsequent to treatment with this hypertonic 
sea-water, those eggs which happen to develop as far as 
the larval stage, never become top-swimming forms. 
Finally, if sea-urchin eggs are in best physiological condition 
at the time of fertilization, close to one hundred per cent, 
of them reach the larval stage; on the other hand, with the 
osmotic method of initiating development, the percentage 
of developing eggs lies far below that obtained from fer- 
tilized eggs of the same female; if the worker is careless, 
using eggs which are not in best condition, he may obtain 
no developing forms. 

Because Loeb noted that the vitelline membrane did not 
separate, and that the larvae failed to swim at the surface 
of the sea-water, he next endeavored to find a method which 
would overcome these deficiencies. The result of his 
investigations was the so-called improved method of arti- 
ficial parthenogenesis for sea-urchins' eggs. This is the 
famous fatty acid plus hypertonic sea-water method, some- 
times called the lysin-corrective factor method. In this 
method the fatty acid mostly employed is butyric acid. 
Loeb first studied the eggs of the California sea-urchin, 
Strongylocentrotus purpuraHis. He placed unfertilized eggs 
in a solution of 50 cc. of sea-water plus 2.8 cc. of J 10 normal 
butyric acid and left them in this solution for one and one- 
half to two and one-half minutes. On removal of these eggs 
to normal sea-water, they separated membranes. Eggs 
removed earlier than the minimum time did not separate 
membranes nor did those eggs which remained in the acid 
solution longer than optimum time, because prolonged 
exposure to the acid is injurious. After the eggs had been 
in normal sea-water for fifteen to twenty minutes, they were 
immersed in a solution of 50 cc. of sea-water plus 8 cc. of 



2.5 gram molecular NaCl. From this solution, beginning 
thirty minutes after immersion, they were transferred to 
normal sea-water at intervals of either two and one-half 
or five minutes. This double treatment of fatty acid and 
hypertonic sea-water results in a development that closely 
approximates the normal, because the eggs not only show 
separated membranes, but the larvae swim at the surface 
of the sea-water. It is to be especially emphasized that in 
this method the order in which the means are used is of no 
consequence to the results obtained: the butyric acid may 
be used before or after the hypertonic sea-water. 

For the eggs of Arbacia at Woods Hole, the method is 
somewhat different. According to Loeb, 2 cc. of J-'io normal 
butyric acid in 50 cc. of sea-water acting from one and one- 
half to three minutes must be employed. Even so, Loeb 
did not succeed in inducing the eggs of Arbacia to separate 
their vitelline membranes; they only showed what he called 
a fine gelatinous layer which was not easily visible. It 
remained for Heilbrunn 1 to show that the butyric acid 
treatment for this egg must be shortened; then the vitelline 
membranes are separated in much the same form as from 
fertilized eggs. This I have confirmed for the eggs of 
Arbacia. Also, I have obtained with butyric acid perfect 
membrane-separation in the egg of another echinid, 

Of this resume, although it is by no means complete, we 
can make the following summarized statement which holds 
for all cases of parthenogenesis experimentally induced in 
marine eggs. 

1. Only when they are in their normal fertilizable period 
do eggs respond to experimental means. 

2. For all eggs, one means only — as heat, cold, acids, 
hypotonic sea-water, hypertonic sea-water, etc. — is suffi- 

1 Heilbrunn, 1915. 

2 Just, 1919c, 1920. 



cient. We shall soon learn that eggs of sea-urchins which, 
with the above discussed osmotic method, develop abnor- 
mally, are no exception to this rule. 

3. Hypertonic sea-water is most generally effective 
although the result is not the same qualitatively for each 
species of egg. 

4. No experimental means is limited in its action to eggs 
of one fertilization-class only. 

No. 4 requires comment. If we arrange eggs into four 
classes, on the basis of their fertilization-moment, we do not 
find that for each of these classes one special treatment is 
alone successful. Heat, for example, is as successful on 
the egg of Nereis in the germinal vesicle stage as on that 
either of the worm, Chaetopterus, or of the clam, Cumingia, 
both in the stage of first maturation. On the other hand, 
eggs of the same fertilization-class do not always respond 
to the same means. Hypertonic sea-water does not induce 
cytoplasmic division in the egg of Nereis, but does in the 
egg of Mactra, which like that of Nereis is fertilizable in 
the germinal vesicle stage. Acid-solutions in sea-water fail 
to initiate development in the egg of Nereis; they call forth 
development in the egg of Thalassemia, fertilizable like 
that of Nereis in the germinal vesicle stage. Moreover, 
consider the egg of the starfish. It responds best when in 
that stage, first maturation, which is optimum for its fer- 
tilization. Carbon-dioxide, butyric acid or heat alone is 
sufficient for eliciting complete response: membrane-separa- 
tion, cleavage and vigorous larvae. Butyric acid alone, 
however, does not initiate development in eggs of other 
species in whatever stage of maturation they are fertilizable. 
Shaking, which suffices for the induction of development of 
the starfish egg, is without effect on other eggs fertilizable 
in the same stage, though in the egg of Nereis it does cause 
the break-down in the ectoplasm and the dissolution of the 
germinal vesicle. 



Thus, as far as we have seen, a single inducing means is 
effective in eliciting complete development in all eggs named 
except in the sea-urchins'. The two exposures to the solu- 
tion of butyric acid in sea-water used on the eggs of the 
starfish are not to be regarded as a double treatment since 
development is initiated by one exposure if it be sufficiently 
prolonged; in the double or intermittent exposure the effects 
are additive. Treatment with two different means, such 
as butyric acid and hypertonic sea-water, is unnecessary 
for all eggs named above, except for the sea-urchin's egg 
which does not develop normally with the original osmotic 
method, as we have seen. 

This point is of fundamental significance since a theory 
of experimentally induced parthenogenesis has been based 
on the exceptional case of the sea-urchin's egg. Upon the 
experience that the double treatment brings about good 
development in the sea-urchin's egg a theory of general 
application was built. And this despite the fact that for 
all other eggs the double treatment is not necessary. But, 
as I was able to prove, also the sea-urchin's egg can be made 
to develop normally by a single parthenogenetic means. 1 

Using a strong solution made up of 20, 22 or 24 parts of 
2}4> M NaCl (or KC1) plus 80, 78 or 76 parts respectively 
of sea-water on the eggs of Arbacia instead of the solution 
employed by Loeb (8 parts 2)2 M NaCl plus 50 parts of 
sea-water), I was able to induce the development of a high 
per cent, of Arbacia eggs. The eggs separate beautiful 
membranes while in any one of these solutions, but need to 
remain in it for some time thereafter to give farther develop- 
ment. 2 On transfer to normal sea-water after the opti- 

1 Just, 1922a. 

2 It should be noted that R. S. Lillie found that to induce complete 
development of the starfish egg- — i.e., to the larval stage — the acid 
must act for a longer time than that sufficient to cause membrane- 



mum exposure, they cleave regularly, later becoming larvae 
which swim at the surface of the sea-water. This method 
is far simpler than the butyric acid plus hypertonic sea- 
water method; and in my experience it is also superior. 
Batallion and Batallion and Tchou working with several 
species of sea-urchins have confirmed my findings. 1 

Thus, the method of double treatment is unnecessary for 
sea-urchins' eggs. They, like all other animal eggs so far 
studied, respond to treatment with a single agent as the 
author of the method of the double treatment himself has 
shown in his original studies on three species of sea-urchins. 

The fact that treatment with a single means initiates 
development of unfertilized eggs renders less difficult the 
approach toward an understanding of naturally occurring 

Every organism, unicellular or multicellular, lives in and 
depends upon a world of its own. Whilst some have all 
the oceans, all the earth, as home, or freely roam all the 
sky, most dwell in a more circumscribed sphere; yet all 
sense quickly changes in the environment that hems them 
in. As with individuals, so with the cells in multicellular 
organisms — to changes in alkalinity, acidity, salinity and 
temperature they are acutely sensitive. Within only nar- 
row ranges of alkalinity, acid-, salt-content and temperature 
can the cells of the human body, for example, exist. 2 Vary 
any one of these factors beyond a certain limit and human 
life becomes impossible, as we all know. The grand wonder 
of the human body is the maintenance of these factors as 

separation. This finding is true for all the marine eggs named. 
The inducing of development in the frog's egg by puncture seems to 
be an exception. 

1 Batallion, 1926; Batallion and Tchou, 1926. See also these 
zuorkers {1933) on Bombyx eggs. 

2 See Barcroft, 1932; and earlier, CI. Bernard. 



Now although other organisms and their cells do not so 
narrowly depend upon environmental changes, as do man 
and other warm-blooded animals, nevertheless they quickly 
respond to changes in their peculiar world, the environment 
to which they are keyed and with which they are in accord. 
For practical purpose, we abstract organisms and cells from 
their surroundings — we could scarcely do otherwise in most 
cases for we can not always study whole organisms or entire 
cells at once but only their parts, each in turn. Still we 
need ever to remember that we indulge in abstracting solely 
for the purpose of comprehension and that an organism or 
cell has a dependent relation to its environment. Life can 
not exist apart from its external world. 

Nowhere, I think, in all biology does this most strong 
relationship, unity, really, of organism or cell and environ- 
ment more forcibly reveal itself than in the problem of 
the parthenogenetic development of the animal egg. The 
problem, how the egg can begin development through the 
effect of some change in its environment, is to us still mys- 
terious. The findings of experimental parthenogenesis 
would help to dispel the mystery if they could serve to indi- 
cate how environmental changes effect the initiation of 
development in naturally parthenogenetic eggs. 

A plausible explanation is suggested by the fact that the 
most commonly used means for inducing parthenogenesis 
are simple changes in the medium, as changes in salinity, 
acidity, and temperature. The normally parthenogenetic 
egg probably differs from that which requires fertilization 
in being more labile and more responsive; as such it would 
react readily to one or another of these most commonly 
occurring changes in the environment. That drying, radia- 
tions, and puncture also induce development may be sig- 
nificant. Certainly, they are environmental changes by 
which experimental parthenogenesis can be induced. Since 
we know these, we should seek to determine whether they 



or others start development in the naturally parthenogenetic 
egg. Until more data are available, we can not go beyond 
such general statements as to the probable cause of natu- 
rally occurring parthenogenesis. 

As to experimentally induced parthenogenesis, however, 
the experimental findings suffice for an attempt at formulat- 
ing a theory. And indeed, many theories have been prof- 
fered. With respect to one of these which became the most 
widely known, one notes a curious, even an anomalous 
situation. Although the data on induced parthenogenesis 
indubitably show that one means alone suffices to call forth 
development in the eggs studied, including those of sea- 
urchins, this theory, whose founder is Loeb, runs counter to 
these findings, including most of its author's, in explaining 
the phenomenon as due to the action of two distinct means 
because of the one fact that with a certain method, eggs of 
sea-urchins develop in more nearly normal manner after 
treatment with two means. 

Since the interpretation of the induction of partheno- 
genesis in sea-urchins' eggs by the double method came 
finally to be the most widely accepted theory of fertilization 
embracing all animal eggs, we must examine it here. In 
this interpretation it is contended that butyric acid or any 
other agent which calls forth membrane "formation" tends 
to destroy, i.e., superficially cytolyze, the egg; and that such 
an agent acts as do haemolytic agents, those that destroy 
red blood cells. The second agent, it is held, corrects the 
destructive action of the first. This interpretation of cer- 
tain experimental findings on eggs of sea-urchins became 
the famous "superficial-cytolysis-corrective-f actor" theory 
for experimental parthenogenesis and for fertilization. The 
question now arises: Do the experimental findings on which 
it is based, actually permit this interpretation? 

Eggs in sea-water, no matter from what species of animal 
they are, eventually die if they are not fertilized, that is, 



they go to pieces, cytolyze. Hence, for the unfertilized egg 
sea-water is normally cytolytic. The length of time which 
eggs can remain in sea-water before they begin to cytolyze 
depends largely upon the temperature of the sea-water and 
the ratio of the volume of eggs to the volume of sea-water; 
also it varies with eggs of different species. 1 Now the fer- 
tilization-capacity of eggs in sea-water diminishes with time; 
here also the factors, temperature and volume of eggs come 
into play. The rate at which the fertilization-capacity 
drops also varies with eggs of different species. But it holds 
generally that the fertilization-capacity disappears before 
cytolysis begins — obviously, it must have disappeared at 
the latest at the moment when cytolysis begins, for a dead 
^gg can not be fertilized. As a matter of fact, the capacity 
of an egg in sea-water to respond to an experimental means 
falls off more rapidly than its fertilization-capacity. This is 
strikingly brought out by the above mentioned effect of 
warm sea-water on the egg of Nereis. Its response to 
treatment with warm sea-water is not so good if it has 
been in normal sea-water before treatment, whilst its 
fertilizability is thereby not lessened. So we consider the 
cytolytic action of the sea-water as the antipode of the 
initiation of development by spermatozoon or by experi- 
mental means. 

By the addition of any one of a number of substances to 
sea-water the rate at which eggs cytolyze can be increased. 
Among these is butyric acid. But the action of butyric 
acid in accelerating cytolysis is by no means invariable. 
On the contrary, the effects which this acid produces depend 
upon its concentration in sea-water and, in the case of 
each concentration which is effective for calling forth mem- 
brane-separation, upon the duration of time that the eggs 

1 In making comparisons of the rate of cytolysis of eggs of different 
species one must be careful that the experimental conditions are 
-uniform in the compared cases. 



are exposed to it. If we designate as optimum the short- 
est exposures which are effective in calling forth complete 
membrane-separation, we find, as my own experiments 
have so clearly established, that sub-optimum exposures 
instead of accelerating, actually retard the cytolysis which 
in time normally takes place in sea-water. 1 Eggs in sea- 
water which show fully separated membranes in conse- 
quence of optimum exposure to butyric acid cytolyze more 
rapidly than unexposed eggs. Over-exposed eggs cytolyze 
at a still more rapid rate. In his experiments Loeb never 
used this reagent properly. That is, he exposed the eggs 
of both the California and the Woods Hole sea-urchins 
beyond the optimum time for membrane-separation. If 
he had learned the best exposure, he probably would not 
have so greatly emphasized in the butyric acid-hypertonic 
sea-water method for the sea-urchin egg the cytolytic action 
of the acid. Basing his theory on over-exposure to butyric 
acid, he created the term, " superficial cytolysis.'' 2 

The superficial cytolysis-corrective-factor theory explains 
the action of the two means for inducing parthenogenesis 
as follows: the fatty acid treatment causes u superficial 
cytolysis" and the hypertonic sea-water treatment follow- 
ing " saves" the egg from this impending death. As I 
stated above, the order in which the two agents are used is 
of no consequence: the exposure of the eggs to butyric acid 
may precede or follow that to hypertonic sea-water. That 
is, the corrective factor (hypertonic sea-water), according 
to the theory, if used first acts to correct where nothing is 
to be corrected and the superficial cytolysis factor (the 
fatty acid) used at the second place, tends to kill the more 
than corrected egg. Thus the sequence in the treatment 
so strongly demanded by the superficial-cytolysis-correc- 

1 Just, 1920. 

2 See Just, 1919c, 1920, 1922a, 1922b, 1930c. 



tive-factor theory not only is not supported by fact but is 
contradicted by it. 

We may dismiss the superficial cytolysis-corrective 
factor theory of parthenogenesis for the following reasons: 
All eggs including those of sea-urchins need treatment only 
with a single means. Further, for sea-urchins' eggs, the 
cytolytic effect that the theory stresses, results from over- 
exposure to the acid. Finally, the fact that the order of 
treatment in the double method can be reversed, makes the 
theory even for the single egg of the sea-urchin untenable. 

These obvious failures of the theory however did not pre- 
vent the exaggeration of its significance to an unusual 
degree. The discovery that eggs could be induced to 
develop without the spermatozoon gave rise to extravagant 
claims by experimental embryologists themselves and 
aroused fantastic notions among laymen. Many hailed it 
as a demonstration of the creation of life. As if the unfer- 
tilized egg were not alive ! According to one report, to 
create life is easy since the means for inaugurating the life- 
process are at once simple and close at hand: one needs only 
two chemicals, vinegar and salt, found in every kitchen, to 
start the egg on its long and complicated journey of develop- 
ment, the end of which is the adult form. The occurrence 
of natural parthenogenesis in the meantime was given scant 
attention; indeed, in some quarters it was entirely over- 
looked. The one fact alone, that nature dispenses with 
spermatozoa for the development of certain eggs — those 
of rotifers, aphids, bees and others — this recognized 
fact of natural parthenogenesis that we have known for 
years, should have saved us from making excessive claims 
concerning the significance of experimentally induced 

As soon as the superficial cytolysis-corrective factor 
theory for experimental parthenogenesis had been elab- 
orated, the attempt was made to use it as an explanation for 



the phenomenon of fertilization. The problem seemed to 
be especially easy, since the double treatment — and, there- 
fore, the factors in the explanation — in these experiments on 
inducing parthenogenesis, appears to have parallels in the 
two phases in fertilization, the external and the internal 
phase. T shortly review these here. 

All eggs respond to insemination, as was pointed out, 
by some kind of surface or ectoplasmic changes which pro- 
duce in some cases very striking results, as the separation of 
the vitelline membrane in sea-urchins' eggs or the extrusion 
of the superficially located material in eggs of the genus 
Nereis which sets as a jelly in the sea-water. Underlying 
these changes is always a disintegration of the superficial 
cytoplasm upon which follows normally a rapid reconstitu- 
tion of the surface. This is the so-called first or external 
phase of the fertilization-process. 

Once within the egg, the nucleus of the spermatozoon 
moves toward the egg-nucleus. Both division-centres arise 
near one or the other of the germ-nuclei or one centre 
near the egg- and the other near the sperm-nucleus. 
The mitotic spindle is established and first cleavage is 
initiated. These phenomena constitute the so-called second 
or internal phase of the fertilization-process. 

With respect to these two phases, it is true that experi- 
mental parthenogenesis in the sea-urchin egg initiated by 
butyric acid and hypertonic sea-water resembles the fer- 
tilization-process inasmuch as the acid calls forth mem- 
brane-separation and the hypertonic sea-water initiates the 
formation of an amphiastral mitotic figure. Therefore, 
the superficial cytolysis corrective factor theory though it 
can not be upheld as explanation of experimental partheno- 
genesis, may, one might think, nevertheless explain fertili- 
zation. Attempting such an explanation, Loeb says that 
the "fertilization by the spermatozoon perhaps depended 
not upon a single chemical agent, but upon a combination 



of two or more 1 which were only fortuitously combined in 
the spermatozoon 57 (Italics are mine). According to him 
it is a fact which he has proved that "the membrane forma- 
tion by the spermatozoon is caused also by a cytolytic 
agent — a lysin." 

I have already pointed out that for the sea-urchin's egg 
Loeb used butyric acid either too long or in too great con- 
centration; such over-exposures very greatly increase 
the rate at which this egg cytolyzes in sea-water. 2 The 
optimum exposure to butyric acid, which for the egg of 
Arbacia Loeb never knew, does not cause this hastening of 
cytolysis. Moreover, neither Loeb nor any one else has 
proved the presence of a lysin or of a " fortuitously com- 
bined combination ' ; of chemical agents in the spermato- 
zoon. Loeb's so-called proof of a lysin in the spermatozoon 
is fantastic and wholly specious. 

In calling forth membrane-separation the effect of butyric 
acid is, to be sure, somewhat similar to that of the sperma- 
tozoon. But there is never membrane-separation in the 
butyric acid solution; only after the acid is washed away 
by bringing the eggs into a large volume of sea-water does 
membrane-separation take place. There is thus an essen- 
tial difference between the immediate action of the sperma- 
tozoon in calling forth membrane-separation and the 
delayed effect of butyric acid. This point raises a further 
question. Since according to the author of the superficial 
cytolysis-corrective factor theory of fertilization, butyric 
acid is only an example of a large class of cytolytic (and 
haemolytic) agents, we must ask if the action of butyric 
acid is, as we should expect and as the author of the theory 
claims, characteristic of the class. 

1 The "or more" I think is added as a margin of safety since the 
theory demands only two. 

2 Just, IQ20. 



Among the cytolytic (and haemolytic) agents named by 
Loeb are i}4 M NaCl and distilled water. Now I find that 
either of these induces membrane-separation in Arbacia- 
eggs while the eggs are in the solution. 1 In a pure 2^2 M 
NaCl solution the membranes separate with extreme rapid- 
ity, 2 so that to follow the process one must use a less con- 
centrated solution. The separation of the membrane in 
hypertonic sea-water is due to the rapid shrinkage of the 
egg-plasma. In distilled water, on the other hand, the 
eggs distend as water enters but the elastic vitelline mem- 
branes distend more rapidly than the plasma so that in five 
seconds the membranes are off the eggs, at which time the 
plasma shows no sign of disruption. With prolonged expos- 
ure the egg-plasma swells and reaches the membrane; 
cytolysis follows. The action of butyric acid in causing 
membrane-separation resembles neither that of hypertonic 
nor that of hypotonic sea-water. In my experience all of 
the cytolytic agents named by Loeb fall into two classes: 
those that do not call forth membrane-separation directly 
but only after they are washed away by transferring the 
eggs to normal sea-water; and those that do act directly, 
by producing either shrinkage of the egg-plasma or exten- 
sion of the elastic membrane. If the spermatozoon's first 
duty to the egg be to inject a lysin, which of the three 
kinds of cytolysis — that of butyric acid, of hypertonic sea- 
water or of hypotonic sea-water — does this behavior of the 
spermatozoon resemble? 

An agent which induces membrane-separation in the sea- 
urchin's egg can not act as a fatty acid, a concentrated neu- 
tral salt solution and distilled water at one and the same 
time. If we consider all the cytolytic agents which have 
been employed, we fail to discover in their mode of action 

1 Just, 1922a, 1928c. 

2 J ust , unpublished observations. 



any one common factor; they act either as butyric acid, as 
strong hypertonic sea-water or as distilled water. That 
is, the cytolytic agents lack a specific factor, whereas the 
fertilization-reaction reveals a high degree of specificity, 
borne equally by the components, egg and spermatozoon. 
If one adheres to the lysin-corrective factor theory of fer- 
tilization, one must assume the addition of a specific factor 
in the spermatozoon imposed upon the nonspecific ''for- 
tuitously combined combinations" of factors, resembling 
butyric acid, hypertonic sea-water and distilled water. 
From this point of view specificity would reside only in the 
spermatozoon, the egg playing no part. We are told that 
"the lysins of foreign animals can get into cells by mere 
diffusion, while the lysins of the same species can not get 
into the egg by diffusion. Only through the motile power 
of the living spermatozoon which acts as a carrier can the 
fertilizing lysin of the animal's own species get into the 
egg." 1 Here the author of this theory reveals the failure of 
well-known physico-chemical agents to act as substitutes 
for that "mysterious complex" called the "living sperma- 
tozoon." The high purpose, to transfer the problem of the 
initiation of development of the animal egg from the realm 
of morphology to that of physical chemistry 2 is frustrated 
at a most crucial point. It is, therefore, needless to discuss 
the problem of specificity further in this connection. 

The order of the two phases of the fertilization-process 
is constant in a normal egg; always the external (ectoplas- 
mic) changes come first and the internal (mitotic) come 
second. Never does a normal egg exhibit the phenomena 
of the internal phase before the ectoplasmic changes charac- 
teristic of its fertilization-process have taken place. As 1 
have already pointed out, in the experimental partheno- 

1 Loeb, 1913. 
- Loeb, ibid. 



genetic development of the sea-urchin's egg by means of 
butyric acid and hypertonic sea-water, the butyric acid is 
equally efficient in action preceding or following the treat- 
ment with hypertonic sea-water. This notable difference 
between " chemical fertilization 3 ' and nature's process the 
theory discounts- Though Loeb was the first to know that 
the order of treatment could be reversed, he made no 
attempt to explain this difference from the natural process. 

Sharply visible ectopias mic changes are always pres- 
ent in fertilization but not in all cases of experimental 
parthenogenesis- As we have seen above, the eggs of sea- 
urchins develop with Loeb's original hypertonic sea-water 
method , although without membrane-separation. The 
double treatment is not essential. 1 

Because of these considerations we must flatly dismiss the 
superficial cytolysis-corrective factor theory as an attempt 
to explain fertilization as we have dismissed it as an explana- 
tion of experimental parthenogenesis; it fails not only for 
eggs generally but also for sea-urchins' eggs specifically. 

But the fact remains that parthenogenesis can be experi- 
mentally induced. What is experimental parthenogenesis ? 

If terms mean anything, we should expect that in experi- 
mental parthenogenesis, as in natural, perfect and complete 
development should result- Yet, as we have seen, generally 
experimental parthenogenetic development does not extend 
beyond the larval stage. This is undoubtedly often due to 
our failures to overcome the difficulty of rearing animals to 
the adult stage from eggs under laboratory conditions. 2 
Some difficulty also inheres in the methods employed: 
means of experimental parthenogenesis often tend to do 

1 It is curious that Loeb insisted that the superficial cytoly sis factor 
is the essential one; he thereby denied his own discovery and deprived 
himself of all ground in his whole fight concerning priority. 

2 / have been able to carry Platynereis megalops through three 
generations under laboratory conditions. 



too much — though they initiate development, they also 
impair it. 

Even if we in time overcome these difficulties, there 
still remains doubt that the action of the means of induced 
parthenogenesis is identical to that of the spermatozoon. 
For while normal, fertilized eggs do, if properly handled, 
give at least 95 per cent, perfect larvae, it is difficult if not 
impossible to obtain this percentage in experimental par- 
thenogenesis. The spermatozoon is more effective than 
a means of experimental parthenogenesis; frequently one 
finds that a certain lot of eggs, a sample of which will give 
more than 95 per cent, fertilization and perfect develop- 
ment, will not respond to a means of parthenogenesis that 
is usually effective on eggs of this species. 

On more general grounds it is still to be doubted that an 
experimental means duplicates the action of the normal 
stimuli in the intact organism, though experimental 
imitation is often possible. Despite these considerations 
however, it must be clearly emphasized that complete 
development can be induced experimentally. 

Even if we do not yet succeed in establishing the par- 
ticular way in which either spermatozoa or experimental 
means act upon the egg so that it develops, we certainly 
can agree that either spermatozoon or experimental means 
in initiating the development of the egg produce the same 
result — a succession or rhythm of mitoses and cleavages 
which finally lead to embryo-formation. As we have 
already seen, the experimental means are not specific. 
Most probably, the nature of the reaction between experi- 
mental means and egg differs also from that between sper- 
matozoon and egg, since there is evidence to indicate that 
fertilization is a chemical union of an egg-substance with 
the spermatozoon, whereas we can assume that the initial 
action of the experimental means is physical. But the 
end-result, however reached, is the same. The conclusion 



is therefore this: the egg-cell like many another living cell- 
nerve or muscle, for example — possesses independent irri- 
tability. It has full capacity for development. Neither 
spermatozoa nor experimental means furnish the egg with 
one or more substances without which the initiation of 
development would be impossible. 

Here lay at the same time the possibilities and the failure 
of the work on experimental parthenogenesis. Every 
single investigator who erred in "proving" an external 
agent (or agents) to be the cause of development neglected 
an opportunity to extend our knowledge concerning that 
fundamental manifestation of living matter, its independent 
irritability. Some theories of fertilization derived from 
work on experimental parthenogenesis postulated as the 
cause of development a change in the egg, e.g., membrane 
separation, that was however merely one consequence 
of the ectoplasmic changes, or they related the cause of 
development to some concomitant of mitosis. Others, as 
had the Loeb theory, merely offered a substitute for the 
sperm-borne centrosomes. Instead of these two structural 
entities which Boveri had for some time postulated to be 
indispensable for fertilization because he thought them to 
be either lacking in the egg or present in too enfeebled state, 1 
the new school of biology substituted all kinds of means, 
like lysins, mysticisms no less mystical because appearing 
to belong to the realm of physical chemistry, having the 
living condition of the spermatozoon always conveniently 
at hand as a refuge finally to be sought. 2 

I do not wish by seeming to dwell over long on this point 
to take unfair advantage of the patent shortcomings of the 
work on experimental parthenogenesis. And yet I must 
hold it up as an example of what such a large section of the 

1 See also Vedjovsk\\ 1SSS-92. 
- Loeb, 1913. 



school of quantitative biology has done for us by over- 
reaching facts and drawing unwarranted conclusions as to 
the physical chemistry of vital phenomena. One method 
used in this work is the " substitution of well-known physico- 
chemical agents" for living components. But this substi- 
tution is only that of a well-known physico-chemical agent 
for another one. One need not be in complete ignorance 
of this agent, i.e., the living cell, unless one takes pride in a 
disdain for the knowledge accumulated by descriptive 
studies. Not hindered by this knowledge one can easily 
make great discoveries and, through fecund ignorance, per- 
petuate error. And if, in addition, with a paucity of 
mathematics, physics and chemistry one elaborates mathe- 
matico-physico-chemical theories, then one by no means 
wins what quantitative biology so much desires: namely, 
security of biology, sitting at the right hand of physics. 
Even those who have an adequate knowledge of physics 
and chemistry and do appreciate the biological phenomena 
which they aim to explain in terms of physics and chemistry, 
should, (when at that point beyond which their data do 
not warrant conclusions) honestly say: "However much 
we desire to establish life as a mechanism, here our present 
knowledge comes to its limits/ 5 It is sad irony that a 
theory of vaunting mechanistic conceptions had, as its 
basis, work the true value of which lay in establishing the 
fact that the egg as a living cell is self-acting, self-regulating 
and self-realizing — an independently irritable system. For 
the spermatozoon, the theory's mechanistic conception is 
more vitalistic than even the ardent vitalist could desire: 
for it says that the living spermatozoon does what it does 
only because it is alive. 1 

The history of biological research furnishes us with other 
examples which illustrate the short-comings of such physico- 

1 Loeb, I.e. 



chemical approach in setting up a singly studied property 
of living matter as synonymous with the whole complex 
of life-processes. Witness the theories of oxidation, per- 
meability, electrical conductivity, viscosity and the like. 
These all have failed; they leave untouched the cardinal 
problem of biology, the structure of a living thing; they do 
not relate themselves to the organization which distin- 
guishes a living thing from a non-living. Or take the almost 
universal fashion in which Hill's work on nerve-conduction 
was accepted. By "proving" that a nerve conducts without 
heat-loss this work must logically lead to the conclusion 
that the nerve-fibre is not living since it gives no evidence 
of metabolism. Strictly orthodox morphologists have like- 
wise often presented theories of life-processes on the basis of 
demonstrations that only emphasize anew the capacity 
of the living thing though debased to respond according to 
its specific and intrinsic irritability. The now perfect 
collapse of the organizator theory is a case in point: all 
that remains of it is a name for the well-known power of 
protoplasm to respond in the same characteristic, struc- 
tural and physiological manner to diverse stimuli. 1 

True, terms, as inherent irritability and response to 
stimuli, are more general than any one denominating a 
physico-chemical attribute named above, or than the term, 
organizator, even. But the present state of our knowledge 
of protoplasmic response does not yet permit the use of 
more specific terms with reference to the behavior peculiar 
to matter organized in the living state. The reproach that 
one retards progress by use of too general terms carries no 
serious onus so long as we have not progressed far enough 
to warrant the employment of precise physico-chemical 

1 As an example , cf. Just, 1936c: here an egg responds zvith the 
same structural change to most diverse experimental means. 



Experimental parthenogenesis, a problem far from being 
solved, invites investigations both for its own sake and for 
the implications it possesses for the biology of cells gener- 
ally. In the interest of this research it is necessary to 
define the problem clearly in order that not all kinds of 
observations are considered as belonging to it: Experimental 
parthenogenesis must be defined as the development of an 
egg that has been initiated and carried to at least the 
normal larval stage by an agent other than the living 

In contrast to this clear definition there exists a great 
deal of confusion in the literature on this subject. For 
instance, an agent which produces almost no change in an 
unfertilized egg, as revealed by the egg's response with 
perfect development when inseminated, is often called a 
parthenogenetic agent. Or the so-called parthenogenetic 
agent induces membrane-separation only, stimulating the 
egg to the limited extent that makes fertilization now impos- 
sible. In other cases the changes induced may be injurious, 
yet the egg does not lose capacity to respond to fertiliza- 
tion, though its development is abnormal. Or the pseudo- 
parthenogenetic agent induces cytolysis which begins sooner 
or later, depending upon the agent's toxicity. Whenever 
the changes induced, either because of the nature of the 
agents themselves or the method with which they are used, 
do not lead to development, they should not be called 
parthenogenetic. And certainly, death changes, i.e., cyto- 
lysis, should be considered to be beyond the limits of the 
term, the initiation of development. 

The establishment of the mitotic figure constitutes, as 
we have seen, a definite and reliable index for the comple- 
tion of the fertilization-process. Normally, development 
then proceeds with rhythmical nuclear break-down and 
reformation; and in the majority of eggs this division of 
the nucleus synchronizes with cleavage of the cytoplasm. 



In some eggs, e.g., of insects, the first nuclear divisions ensue 
without cytoplasmic cleavages, whilst in others, e.g., those 
of selachians and of birds, later mitoses proceed without 
the splitting up of the cytoplasm. In certain eggs experi- 
mentally treated, some kind of development, abnormal 
however, can go as far as a swimming form without cyto- 
plasmic cleavage. Hence, although development never 
completes itself without the earlier or later breaking up of 
the cytoplasmic mass of the egg into cells, we can not cate- 
gorically define the process of development as related to 
the sundering of the cytoplasm. Therefore the end of the 
fertilization-process we define by the appearance of the 
mitotic figure rather than by the first cleavage of the 
cytoplasm. 1 

Unfortunately, we have neglected to make nice studies 
on the differences in the development of two sets of eggs 
from the same animal, one set fertilized and the other 
treated with some means of inducing parthenogenesis- 
such differences, for example, that would reveal themselves 
in the size of cells and in the time and the place of their 
appearance in an egg whose cell-lineage is known, that 
is, 'one in which the size, order of appearance and loca- 
tion of the cells with reference to each other are definite 
and invariable. Some differences, as rate of cleavage, we 
know; others, indicated for the earlier stages of develop- 
ment, are doubtless due to the fact that the experimental 
treatment never quite duplicates the action of the sperma- 
tozoon. But even if we assume that fertilized and par- 
thenogenetically developing eggs, both in the induced and 

1 / say mitotic figure because the occurrence of amitotic nuclear 
division in developing animal eggs has been doubted. If toe include 
the possibility of development with amitotsis in our definition, ive 
substitute in the thesis nuclear division for the term, establishment, 
or appearance, of the mitotic figure. 



the naturally occurring process, differ in their course of 
development, we know certainly that by either fertilization 
or parthenogenesis, if development is complete, an adult 
animal emerges through a succession of nuclear and cyto- 
plasmic divisions. Therefore, despite any differences that 
may occur during the developmental process, the beginning 
stage is always that of the establishment of the cleavage 
figure with consequent nuclear and cytoplasmic division, 
the end-stage always being the adult form. The question 
thus arises as to the nature of the calling forth of 
the nuclear configuration through whose subsequent 
rhythmical behavior together with cytoplasmic cleavage 
the end-stage is attained. The initial action of spermato- 
zoon and of experimental means being different, and dif- 
ferences in action obtaining between one means and another, 
the question at issue is: Do these differences persist so that 
the calling forth of the process of nuclear and cytoplasmic 
divisions springs from various causes, or do these means, 
whatever they are, elicit the same reaction which sets 
up the division-process? I suggest that they set up the 
same reaction. 1 Let us briefly review the experimental 

In sea-water sufficiently hypertonic to induce develop- 
ment, eggs of any species which respond to such treatment 
rapidly and directly lose water. The minimum hyper- 
tonicity capable of calling forth development in sea-urchins 5 
eggs does not bring about a sharply defined shrinkage of the 
eggs from their vitelline membranes; only in stronger hyper- 
tonic solutions does this egg shrink from its membrane. 
Other eggs shrink in hypertonic solutions to such an extent 
that the vitelline membranes no longer adhere to the cyto- 
plasmic surface. On return to normal sea-water, the eggs 
take up water but they do not regain the equilibrium with 

1 See Just, 1927a. 



the sea-water which they possessed in their untreated condi- 
tion, instead they establish it at a new and different level. 

In hypotonic sea-water eggs take up water and increase 
in volume. If the degree of dilution is effective, the rapid 
intake of water induces break-down of the eggs' surface- 
cytoplasm and rapid distension of their vitelline membranes. 
Removed to normal sea-water they quickly diminish in vol- 
ume but do not regain their previous state because of the 
complete break-down at the surface, i.e., complete ecto- 
plasmic changes. They come again into equilibrium with 
the surrounding sea-water, but this, owing to the altered 
surface-structure, is at a new level. 

After having been properly exposed to effective acids in 
optimum concentration in sea-water, eggs on return to 
normal sea-water separate their vitelline membranes. That 
is, the acid treatment induces a change which brings about 
the subsequent break-down in the surface-cytoplasm. 
With the processes of restitution by which a new surface- 
layer forms, the eggs come into a new equilibrium with the 

Warm sea-water when effective causes immediate break- 
down in the surface-layer. Puncture of the frog's eggs pre- 
sumably leads also to ectoplasmic break-down. Radiation, 
as of ultra-violet, brings about ectoplasmic break-down. 
In these cases as in the others given above, the surface- 
structure is altered and thus the equilibrium between egg 
and sea-water is different from that which existed before 
the treatment. 

Also after fertilization an egg having undergone complete 
ectoplasmic changes does not return to the state which it 
had before fertilization with respect to its equilibrium with 
the sea-water. It shows instead a marked difference. 
With its altered surface-layer, the fertilized egg establishes 
a new equilibrium with the sea-water. As we shall see in 
the chapter that follows, fertilized eggs exhibit in their 



reaction to environmental influences rhythmical changes of 
resistance and susceptibility that are both different from 
those shown by the unfertilized egg. 

The new egg sea-water equilibrium is established by the 
structural changes in the ectoplasm; these changes do not 
reverse themselves and the egg does not return to the 
physiological state it had previous to treatment or to fer- 
tilization. Also with respect to water-movements these 
same changes sharply set off the devel ping egg from the 
untreated and unfertilized. Since the egg loses and gains 
water concomitantly with each cleavage-cycle, one can not 
say that with break-down of substance in the ectoplasm it 
becomes permanently dehydrated. Rather, a momentary 
water-loss as a consequence of the stimulus of inducing 
means or of the spermatozoon, brings about a change to a 
new level with respect to equilibrium with the surrounding 
medium, and on this new level the ensuing rhythmical 
process of water-entrance and -exit which accompanies the 
developmental process takes place. 

The rhythmical movement of water, during the cleavage- 
cycle, into and out of the egg undoubtedly means a move- 
ment of water from place to place within the egg; and this 
in turn means local and temporary hydrations and dehydra- 
tions. These redistributions of water even in minute intra- 
cellular dimensions are favorable for reactions. 1 One 
visible structure of the cell which in addition to the ecto- 
plasm exhibits rhythmical changes during the cleavage- 
cycle is the nucleus ; its break-down and reformation is 
indeed the criterion used for defining the cleavage-cycle. 
As we have seen in the chapter on the fertilization-process, 
the establishment of the mitotic complex constitutes the 
index of the completion of the initial stage of development. 
And I suggest that in all modes of initiating development, 

1 Just, 1937b. 



that of experimentally induced and naturally occurring 
parthenogenesis as well as that of fertilization, the estab- 
lishment of the first mitotic figure is brought about by a 
dehydration of some constituent of the ground-substance, 
or as the result of a reaction between nuclear and cytoplas- 
mic ground-substance brought about by dehydration. The 
mitotic figure then is either expressly a dehydration-for- 
mation or the result of a reaction rendered possible by 
dehydration. In the resting egg this material in the 
ground-substance is spatially diffused; with the process of 
parthenogenesis or of fertilization it becomes aggregated — 
in parthenogenesis and in some cases of fertilization around 
the egg-nucleus, in most cases of fertilization around the 
sperm-nucleus. What we see as asters and as spindle may 
be either this material itself or the sign or product of its 

This hypothesis — and more can not be proffered in the 
present state of our knowledge — is wholly consistent with 
the established facts as far as one can reduce them to order. 
According to it the most important factor in all modes of 
initiating development is a dehydration-process affecting 
directly or indirectly the ground-substance. 1 In all cases 
of the initiation of normal development, dehydration begins 
in the ectoplasm. 

Ectoplasmic changes alone, as I have shown above, are 
not experimental parthenogenesis. They do not of them- 
selves determine that development will follow; but they are 
a reliable indicator for the quality of the development, if 
this ensues. That there are cases of ectoplasmic changes 
without subsequent development on the one side and that 
the sea-urchins' egg after treatment with weak hyper- 

1 Delage in I go lb promulgated a dehydration-theory of fertilization; 
but this differs from the one here proffered in ascribing dehydration 
to the influence of the sperm only. 



tonic sea-water develops without complete break-down of 
ectoplasmic substance on the other, warns us against 
attributing the cause of experimental parthenogenesis 
directly to physical changes at the egg-surface, i.e., to 

Far from being an exception to be explained away, the 
case of sea-urchins 5 eggs in responding to treatment with 
the weak hypertonic sea-water without membrane-separa- 
tion and with poor quality of development reveals clearly 
what other eggs more sensitive in their ectoplasmic response 
to experimental means do not: the ectoplasmic changes are 
significant for the development in prospect. Again, as in 
fertilization, we see that the quality of development depends 
upon the quality of these initial ectoplasmic changes. 

They are violent eruptions which precede the evanescent 
spinning that will accompany cleavages yet to come and 
that will build gossamer-like tendrils to bind cell to cell. 
Spermatozoon or parthenogenetic means, Nature's or exper- 
imenter's, set the life of the egg in quicker motion; the 
mitotic spindle comes and goes. The web of life gives a 
pattern which we come to know as larval sea-urchin, worm 
or clam. But almost with the moment that the egg's 
vital activity moves at a quicker tempo we know through 
the ectoplasmic behavior the quality of the events to come. 





or parthenogenetic egg, a single cell, develops into an adult 
organism which may comprise millions of cells. The first 
division or cleavage separates the egg (in most cases) into 
two portions, blastomeres, which remain attached. Suc- 
cessive cleavages give rise to many blastomeres, the tgg 
becoming a mass of cohering cells. The surface of the 
frog's egg, for example, in late cleavage so closely resembles 
that of a golf-ball that one gains the impression that sub- 
division of its surface by cleavage-planes is the egg's chief 
characteristic. If one examines a smaller egg, e.g., of a 
sea-urchin, under the microscope, one notes again the 
resemblance of its corrugated surface to a golf-ball; looking 
closer, one observes within each blastomere a nucleus. 
It is a very simple matter to convince oneself by continuous 
observation on a living egg throughout its cleavage period 
that the "golf-ball" has arisen by successive divisions of 
both nucleus and cytoplasm. 

The cells which comprise an adult organism are not all 
alike in structure. To the naked eye a strand of nerve is 
readily distinguishable from a strand of muscle of equal 
length and thickness; and a piece of kidney can not be mis- 
taken for liver. As en masse, so singly, under the micro- 
scope, the cells of nerve, muscle, kidney, and liver are easily 
recognized. Since these cells have different functions, 
development means something more than mere multiplica- 
tion of cells; it implies the cells' differentiation. This 
arises in the process of cell-multiplication at an earlier or 



later stage, depending upon the species of egg. As we shall 
see, this old problem of differentiation is still the major one 
of the study of development. 

With successive cell-divisions during cleavage, the 
blastomeres become progressively smaller. If this process 
were to continue indefinitely, the size of the cells would 
approximate zero and cell-division would come to a stand- 
still. But this never happens, since in later stages the 
cells transform food into protoplasm. In many cases the 
egg soon after hatching as a larva takes in food; or 
the embryo, as that of the chick, utilizes the yolk which 
is present as reserve food material within the egg. 

Generally, growth is increase in size. A cell may grow 
as such by the intake and transformation of food. In the 
initial stages of the development of an egg, cell-multiplica- 
tion precedes without any increase of volume. The total 
mass of the egg of a sea-urchin, for example, at the end of 
its cleavage-period is approximately the same as at the 
beginning. In the larval stage, cell-multiplication follows 
upon the intake and transformation of food by the cells; 
thus here through the combined process of increase in vol- 
ume by the single cells and increase in their number, growth 
of the embryo is attained. 

In the adult organism derived from the egg, cell-division 
operates daily in the restitution process. Cells of the 
human skin are constantly being replaced. Each day the 
blood-forming tissues in bones must throw into the blood- 
stream millions of red blood corpuscles to compensate for 
the disintegration of older ones. Where there is no more 
capacity for cell-division, any injury becomes tragic; thus 
the capacity for regeneration in the human central nerve 
system is meagre and the possibilities of repair after surgery 
slight because its cells have lost the power of division. 
Organs which possess reparative capacity have retained 
the capacity for cell-division. 



Cells in the adult body frequently show a sudden and 
exaggerated burst of division-activity which results in 
abnormal growth. Thus tumors are built up by riotous 
cell-proliferation. The malignant tumor, cancer, in this 
respect is not different from the benign and to this extent 
any work on cell-division may be of significance for the 
cancer problem; this, however, does not mean that every 
work on cell-division in various forms of animals from 
Amoeba to vertebrate has direct bearing on the problem of 
cancer, since here malignancy points to a cause lying beyond 
the facts which can be established for tumors in general, 
both malignant and benign. 

The germ-cells, eggs and spermatozoa, display the most 
intense activity of cell-division, especially during their 
periods of multiplication. A great deal of our information 
concerning cell-division is derived from the study of these 
cells particularly in their period of maturation. 

It is not only among multicellular organisms as egg or as 
adult that cell-division is of consequence; among unicellular 
animals and plants it is often the sole mode of reproduction. 
These single-cell forms live for minutes, hours or days as 
such and then by dividing into two parts bring into being 
"new" individuals. Or one cell may divide into several 
parts each of which becomes a "new" individual. 

In the non-living world, substances may divide, and so 
multiply and also differentiate and grow. But these phe- 
nomena are quite unlike those exhibited by living things. 
Cell-division constitutes a fundamental process which is 
never observed outside the world of living things. And 
yet, it still lacks an explanation to which biologists agree. 
It is, moreover, most often incorrectly defined. The reader 
should understand that in the following pages my main 
purpose is to derive a definition. Only after having done 
this shall I offer an explanation of this phenomenon, the 
division of the cell. 



Before I begin the discussion I should like again to point 
out how necessary it is that we appreciate differences 
normally appearing in biological processes before we 
attempt to evaluate these processes. With respect to the 
process of cell-division, we recognize two kinds. Based 
upon the behavior of the nucleus in cell-division as a criter- 
ion, two categories of cell-division have been set up: that 
with direct (amitotic) and that with indirect (mitotic) 
division of the nucleus. 

In direct or amitotic nuclear division, the nucleus by 
constriction separates into two parts. It first elongates, 
taking the form of a dumb-bell or hour-glass, and then 
breaks into two equal or unequal portions. Each of these 
with cytoplasm constitutes a new cell, if the cytoplasm also 
divides. But division of the cytoplasm synchronously 
with that of the nucleus is not invariable. The occurrence 
and significance of amitosis have been much debated. 
Some few biologists hold that amitosis is more widespread 
among animals, even in the development of eggs, than is 
generally believed and that it may be of equal significance 
with mitosis. The majority opinion is that amitosis is of 
restricted occurrence and when found among multicellular 
organisms is of little significance generally, except as a sign 
of decadence or of too highly specialized cells. 1 For the 
Protista we meet the view that what is often called amitosis 
is a disguised mitosis. Nevertheless, it is generally 
admitted that among both multicellular and unicellular 
organisms amitosis does occur. It must, therefore, be 
embraced by any theory that attempts an explanation of 
cell-division, especially if the explanation relates division 
of the cytoplasm to that of the nucleus. 

As has been pointed out already, mitosis or indirect 
nuclear division involves a series of orderly maneuvers of 

1 Wilson, 1925. 



the chromosomes on a set of "fibres/ 5 the mitotic spindle, 
beginning with the break-down of the resting nucleus and 
ending with the formation of two resting nuclei. If from 
the poles of the spindle where the "fibres" more or less 
converge, other "fibres/ 5 called astral fibres, extend radi- 
ally into the cytoplasm, the spindle is said to be of the astral 
type; if these radiations are absent, the spindle is called 
anastral. In the former type at the centre of the aster may 
be found either a single granule, the centriole or centro- 
some, or many finer granules; or, the astral centre may be 
of such fine structure that it appears to be optically empty. 
Mitotic division of the nucleus may normally ensue without 
concurrent division of the cytoplasm; experimentally it is 
easily possible to suppress or arrest cytoplasmic division 
while nuclear divisions continue. Since this is true, the 
failure of cytoplasmic division after amitotic nuclear divi- 
sion that is sometimes observed, does not deserve the 
emphasis which some writers place upon it. The mitotic 
complex may arise wholly or in part within or outside of 
the nucleus. 

What should we demand of any theory of cell-division? 
An explanation of cell-division and not of nuclear phe- 
nomena. The theory should cover amitosis and mitosis. 1 
It should apply with equal force to all types of mitosis 
among both Protista and multicellular organisms; to every 
variation of the mitotic configuration, i.e., to spindles with 
and without asters, with and without centrosomes. Since 
cytoplasmic division does not always synchronize with 
either direct or indirect division of the nucleus, a theory of 
cell-division based exclusively or too strongly on nuclear 
division embraces only cells exhibiting synchrony in division 
of cytoplasm and nucleus; not only does such a theory fail 

1 Delage, iSg§^ p. 758: u Ce qui est essentiel dans la division indi- 
recie, c* est la division directe et cette derniere seule est a expliquer" 


to account for cells lacking this synchrony, but it implies 
that they are abnormal. The synchrony of nuclear and 
cytoplasmic division is an interesting phenomenon but not 
an essential characteristic of cell-division; instead of being 
over-emphasized in theoretical considerations, it needs 
itself to be explained. 

A theory devised to explain division of the cell-body 
should also be consonant with the observable phenomena in 
the cell both whilst living and when properly fixed. Phy- 
sico-chemical speculations however ingenious when incon- 
sistent with the observed phenomena lend little to the 
solution of the problem. Indeed, we shall notice that in 
the attempt to explain cell-division the ignorance of the 
actual process and the exaggerated application of physico- 
chemical knowledge have together erected serious obstacles 
to a proper understanding and that, as the research on 
experimental parthenogenesis, so also that on cell-division 
can be upheld as an example of the danger which inheres in 
this kind of physico-chemical study. Again it should be 
emphasized that the first task in the approach to the prob- 
lem is to learn as thoroughly as possible the normal process 
which is to be explained. 

Unfortunately, explanations of the mechanism of cell- 
division have frequently been based on abnormal cell- 
behavior induced by experimental agents. The chief error 
in this type of work does not lie in the use of a strong experi- 
mental means that brings about abnormality; it lies in the 
fact that the conclusions overlook this result of the treat- 
ment. Further, it is clear that experiments, in which the 
cells were weak and abnormal at the outset, can not serve 
as a basis for the explanation of the normal process. 

For a theory of cell-division, as for any problem in bio- 
logy, an appreciation of the normal biology of the cells under 
their natural conditions is imperative. For example, 
important as the work on vertebrate cells in tissue-culture 



is for that on cell-division, we must always reckon with the 
fact that these cells are not in their natural medium and 
also that they have escaped the integration under which 
they live while in the intact organism. Similarly, it is 
dangerous to interpret the mechanism of cell-division from 
the study of living cells after these have been dissected out 
of the organism. Those eggs normally shed and insemi- 
nated in the sea and free-living protozoa offer the best 
opportunities for observations on cell-division in the living 
cell under conditions approximating the normal. Since 
here we deal with metazoa, I concentrate the discussion 
on them, restricting myself largely to animal eggs. This 
I do, not only because this book concerns itself with animal 
eggs, but also because the most prominent theories on cell- 
division are based on the study of eggs, especially those of 
the sea-urchins. Nevertheless, we finally encompass cell- 
division in all animal forms. 

Obtainable in large number, easy to handle, of convenient 
size and comparatively simple in structure, eggs of various 
species of sea-urchins have been most popular cells for the 
study of cell-division. The cytoplasm of some is trans- 
parent or nearly so and in them one clearly discerns in the 
successive stages of nuclear division the behavior of the 
various constituents of the mitotic complex, including 
the chromosomes once they appear on the spindle; in others 
because some cytoplasmic granules are brightly colored, 
one marks by their shift the ebb and flood of the cytoplasmic 
tides. The study of the pigmented type supplements that 
of the transparent. Also, the transparent eggs one can 
color by means of inert dyes that do no harm to the living 
cytoplasm, and can in the pigmented ones by innocuous 
centrifugal force mass the colored granules so that the 
nuclear phenomena more sharply stand out. But the cells 
must be normal; when fertilized they should be of the same 
specific gravity and almost perfect spheres, all showing 



subsequently at each instant in the division-process the 
same change in contour and exhibiting a clock-like precision 
in the cleavage-rhythm. 

Another advantage inheres in the use of sea-urchins' eggs 
for the investigation of cell-division. Because of the fact 
that these eggs are fertilized after complete maturation, the 
process of division is most easily observed, being that of a 
single division-cycle and not imposed upon the events of 
one or both maturation divisions as in eggs of the other three 
fertilization-types. Moreover, the cleavage is of the most 
simple type inasmuch as the first three cleavages form 
almost equal and close to spherical cells. Since the first 
cleavage cycle encompasses the union of the egg- and sperm- 
nuclei while all succeeding cleavages are wholly mono-nu- 
clear — a fact which we must ever bear in mind — it would 
appear necessary to appreciate the sequence of events in 
the second or the third cleavage in order properly to evalu- 
ate those in the first in which part of the events belongs to 
the fertilization-process. One must be fully cognizant of 
what events belong to fertilization and what to cell-division. 
To obtain a clear picture of the end of the one and the 
beginning of the other process is not easy since so much of 
the work on cell-division considers the first division only. 

The account now given is based upon the egg of Arbacia 
because of its widespread use by investigators, and not 
upon the more favorable but more sensitive egg of Echi- 
narachnius, equally familiar to me but not so extensively 
employed by others. My experience with these eggs as 
well as with those of four different species of sea-urchins at 
Naples convinces me that, despite certain structural dif- 
ferences and some variations in their response to experi- 
mental treatment — due to habitat, as, for example, 
temperature and salinity of the sea-water — what I here set 
forth applies generally to echinid eggs. In order to make 
comparisons with the work of others who followed the 



events of the first cleavage-cycle only, my account will be 
limited similarly. This record of the happenings in the 
living egg is supplemented by that obtained through study 
of the fixed egg. 

Let us begin the account of the first cleavage-mitosis in 
the egg of Arbacia with the stage when the two germ-nuclei 
have disappeared by complete fusion to form a single resting 
nucleus, a clear vesicle almost spherical in shape lying in the 
polar axis just above the equator of the egg. At this 
moment two centrospheres with asters are present at oppo- 
site poles of the nucleus; the egg is spherical, oil-drops and 
yolk-spheres, lying outside of the spindle-area, are evenly 
dispersed, the pigment granules are trapped at the egg- 
surface and the hyaline plasma-layer is clearly defined. 

In the process of cleavage we shall follow especially these 
changes: the growth of the centrospheres and asters, the 
varying shape of the egg, the shifting of the cytoplasmic 
inclusions and the activity of the hyaline plasma-layer as 
the nucleus goes through its mitotic cycle. These four 
changes are easily visible and readily followed in the living 
egg. In accordance with the principle hitherto followed, I 
shall endeavor to derive a conclusion as to the meaning of 
the process by relating the events that can be observed. 

The nucleus seen in the living egg does not retain its 
spherical form; increasing in size it becomes ellipsoid and 
ruptures at the poles of its long axis where the centrospheres 
and asters are located. A row of hyaline droplets, the 
chromosomes, extends across the equator of the transparent 
ellipsoid nuclear region; soon these form two parallel rows 
which slowly separate as the ellipsoid nuclear region 
by a constriction at the equator becomes dumb-bell 
in shape. As each group of droplets approaches the inner 
border of the centrosphere (the centrospheres are now hemi- 
spheres presenting their plane-surfaces toward the dumb- 
bell shaped clear region) they become spherical. Suddenly 



they are in the centrospheres where they fuse to form 
five or six droplets; these by farther fusion become one 
hyaline mass, the resting nucleus. 

Concomitantly with this behavior of the nucleus and the 
movement of the hyaline droplets, the centrospheres 
steadily increase in size and the astral rays become more 
extensive until the moment that the hyaline droplets enter 
the centrospheres. Thereupon the centrospheres diminish 
in size and the astral rays lose definition and shorten. Dur- 
ing their period of growth the centrospheres are spherical; 
their change to hemispheres marks the beginning of decrease 
in size. 

In the meantime the egg exhibits changes in shape. 
While the hyaline droplets are moving toward the poles of 
the transparent dumb-bell shaped area, the egg maintains 
its original spherical form. With the entrance of the hya- 
line droplets into the centrospheres, when centrospheres and 
asters have begun to wane, the egg quickly elongates in the 
long axis of the transparent region. About one minute later 
the egg divides by a furrow forming at right angles to the 
egg's long axis. Thus the egg at first a sphere becomes an 
ellipsoid which divides and then becomes two spheres. 

The site of the future cleavage furrow is marked as early 
as the stage in which the transparent ellipsoid area is con- 
verted into a dumb-bell shape. Already moved by the 
fertilization-reaction, the various cytoplasmic inclusions, 
especially the yolk, are further distributed by the growth 
of the sperm-aster; and later, by the formation and exten- 
sion of the amphiastral system. The conversion of the 
ellipsoid area to the dumb-bell shape is caused by the move- 
ment of yolk-spheres in the equatorial plane of the egg 
from the periphery toward the centre. The shifting of 
the oil-drops can not be easily followed in the living egg. 
The pigment granules, after fertilization definitely fixed to 
the outer rim of cytoplasm, at the time of cleavage extend 



across the egg in two parallel rows, one on each side of the 

Concurrently with these nuclear and cytoplasmic events, 
the clear peripheral cytoplasm, the hyaline plasma-layer, 
exhibits changes. During the progress of the sperm- 
head toward the egg-centre, the hyaline plasma-layer 
is everywhere of equal width; but with the beginning of 
the cleavage-process it appears delicately crenated, irregular 
in contour. The egg at this time being somewhat darker 
because of closer approximation of the yolk-spheres gives 
the picture of cytoplasmic contraction, a condition which 
passes leaving the egg clearer and the hyaline plasma-layer 
of smoother contour. Immediately prior to the egg's 
elongation, the hyaline plasma-layer is thinner over the 
poles of the long axis of the transparent dumb-bell shaped 
area and seems slightly thicker at the equator, the site of 
the future cleavage-furrow. 1 With cleavage this thickening 
apparently increases; actually, the filaments of the hyaline 
plasma-layer are stretched, because the plasma-membrane 
to which they are attached does not move inward with the 
cleavage furrow; they are therefore longer here than 

We can confirm and extend these observations on the 
living egg by study of properly fixed normal eggs. The 
accompanying figures are from sections of fixed Arbacia 

Fig. 33*2 is of an egg with intact nucleus, centrospheres 
and asters. Fig. 3 3 Z? shows an egg with ruptured nucleus 
and emerging chromosomes (seen in the living egg as hya- 
line droplets); here the centrospheres are larger and the 
astral rays more extended. As the heavily stained chro- 
mosomes move apart, the centrospheres and asters increase 
in size (Figs. 33*:, 33^) finally to reach the maximum 

1 Cf. Ziegler, 1904. 



Fig. 33. — For decriptive legend see page 263. 



FlG - 33-~ for decriptive legend see page 263. 



Fig. 33. — For decriptive legend see page 263. 


Fi c 33— For deceptive legend see page 263. 


* « * * « V* - A \ 

* » 


Fig. 33. — For decnptive legend see page 263. 



Fig. 3}. — Stages in the first cleavage-cycle of the egg of Arbacia. (Drawn by 
Mrs. L. G. Hugueley from author's preparations). 

(Fig. 33*). As the variously shaped chromosomes are 
converted into lightly stained spheres (Fig. 33/), they 
are at the inner border of the now no longer spherical 
centrospheres; the wide-flung astral rays are losing defi- 
nition. When the chromosomes within the centro- 
spheres begin to fuse, centrospheres and asters are on 
the wane (Fig. 3 3g). A moment later the egg elongates 
(Fig. 33A); cleavage quickly follows (Fig. 332, j, k) to give 
rise to two spheres. 

For the sake of simplicity and in order that the reader 
may not be confused, I omit at this place a description of 
the evanescent changes in the hyaline plasma-layer. 
Indeed, the figures that I have given here do not show 
the layer. A structure concerning which biologists are at 
odds, because of lack of knowledge, ought be set off from 



the discussion involving better known structures. The 
changes in the hyaline plasma-layer are therefore taken up 
beyond, after its structure has been elucidated. 

The foregoing description of the changes in nucleus, 
centrospheres and astral radiations agrees with the accounts 
given by others for the eggs of several species of sea-urchins. 
According to all of these the centrospheres and astral rays 
steadily increase in size until the stage of the telophase 
whereupon they begin to decrease; later, cleavage occurs. 
That is, cleavage does not take place when centrospheres 
and astral rays are at their maximum size. In spite of this 
mass of well established observations, theories of the 
division of the cell-body have been fabricated upon the 
false idea of a coincidence of cell-division with the maximum 
size of the aster. 1 I do not see any necessity to discuss 
such theories. 

As stated above, this chapter, whilst it deals with cell- 
division primarily in eggs, has to do with the process in its 
widest aspects. Thus we are here concerned with all forms 
of division of the protoplasmic mass. It is to the phenome- 
non as one widely occurring in all living cells that I wish 
now to direct attention. I raise the question: Do the phe- 
nomena observed in the division-process of sea-urchins' 
eggs possess general significance for the problem of cell- 
division ? In order to answer this question Ave must estab- 
lish those features in the division of the sea-urchins' eggs 
which have their parallel in the division both of eggs of 
other species and of other cells, and must eliminate whatever 
is peculiar to this egg only. 

There are other eggs that like the sea-urchins' show that 
form of mitotic division of the nucleus in which centro- 
spheres and asters are present. But whilst the centro- 
spheres of the first cleavage-figure of eggs of sea-urchins 

1 Chambers, 1924; Gray, fpji. 



lack centrosomes, as we have seen in the chapter on the 
fertilization-process, other eggs, indeed most animal eggs, 
possess them. The reason for this difference is here beside 
the point. The difference exists and its existence warns 
us to be wary in drawing too final and dogmatic conclusions 

a b c 

Fig. 34. — Cell-division, living cells of Triton (after Peremeschko). Note changes 

in the ectoplasm. 

with respect to these components. Moreover, were we to 
compare the first cleavage-stage in other eggs with that in 
sea-urchins 5 , we should find no strict correlation of the 
appearance of the cleavage-furrow with either the phase of 
mitosis or the size of the centrosphere-aster complex. When 
the egg of Ascaris, for example, 
divides, the chromosomes in 
early telophase are still strongly 
stained; no such pronounced 
centrospheres as in sea-urchins' 
eggs are present. Briefly said, 
furrowing of the cytoplasmic 
mass of eggs does not occur 
always at precisely the same 
stage of mitosis in all species 
of them. Nor does it occur 
uniformly in other animal cells. 
(Note for example Fig. 34 and Fig. 35.) This is thus my 
first point: We must be sure by exact observation as to what 
happens in sea-urchins 5 eggs; but with this knowledge we 
are not justified in drawing conclusions concerning division 


a b 

Fig. 35. — Stages in cell-division, 
spermatogenesis of Pyrrhocoris aper- 
tus (after Gross). 


in cells generally, if we can base these conclusions only on 
the nuclear behavior and state of sea-urchins' eggs. 

Besides, as I stated above, many cells lack asters, many 
others lack well-defined centrospheres though they possess 
asters. Certain eggs, such as those of Ascaris, of Ctona, 
of Phallusia, of Amphioxus, etc., have no asters at the 
spindle-ends in the maturation-divisions. Yet they divide. 
Many plant-cells go through mitotic division; their mitotic 
figures are without asters. Asters and centrospheres are 
not essential for mitotic divisions. 

Also we should not forget that in many eggs repeated 
mitotic divisions ensue without cleavage of the cyto- 
plasm. Take eggs of insects, for example: many nuclear 
divisions ensue before the cytoplasm divides. Nuclear 
division without cleavage of the cytoplasm is not limited 
to eggs. It obtains among unicellular animals and among 
multicellular, including man, and among plants, in such 
protoplasmic systems that contain many nuclei, the syn- 
cytia. That nucleus and cytoplasm may divide a-syn- 
chronously constitutes the strongest evidence against the 
proposition that in mitosis lies the cause for the sundering 
of the cytoplasmic mass. 

Finally, an explanation of cell-division founded upon the 
mitotic complex as a whole or upon any component of it, 
falls to the ground for it can not encompass division of 
cells whose nuclei divide amitotically. Whatever the 
biological significance of amitosis, be it a primitive or a 
degenerate process, undubitable cases of amitosis are on 
record. Whatever the conditions responsible for its occur- 
rence, we must reckon with this as fact. Even if the 
mitotic complex were always uniform and cell-division 
with mitosis were identical in all cells, we still could not 
explain by reference to mitosis the division of the cell-mass 
where amitosis prevails. 



It becomes obvious in the light of what has been said 
that nuclear and cytoplasmic division are separate phe- 
nomena. 1 Common usage has been loose in giving the term, 
cell-division, the meaning of the division of the nucleus. 
The statements made above, however, emphasize that we 
must define cell-division more clearly and more exactly in 
order to avoid the building up of theories on improper 
grounds. Cell-division is to be defined as the division of 
the cell-body. In what follows I shall adhere strictly to 
this clear definition. 

Finding it impossible to relate division of the cytoplasmic 
mass to the nucleus, we turn to the cytoplasm itself. 2 Here 
we may recall three visible events, accompanying the 
division-cycle in eggs: changes in shape of the egg, move- 
ment of the cytoplasmic inclusions, and changes in the 
behavior of the hyaline plasma-layer. The second and 
third are not peculiar to eggs and will be discussed in detail. 
Change in shape not being invariable is not given great 
emphasis apart from its being a factor in surface-tension- 
theories of cell-division taken up beyond. I speak first of 
the movement of the cytoplasmic inclusions. 

Movements in the cytoplasm constitute a widely occur- 
ring phenomenon, some cells exhibiting them to an extreme 
degree. The simplest type is Brownian movement, so 
extensively studied by physicists in inanimate systems. 
This can be beautifully demonstrated in many cells espe- 
cially by means of the darkfield in which the suspended 
particles appear as bright points which shimmer on the 
black background. Quite different from this phenomenon, 
which is not peculiar to living cells, are the cyclical move- 
ments in cells — often described for plant cells — a streaming 

1 Waiase, iSqi. 

2 Cf. Delage, 1895, P- 759- 



that always follows a definite path or along preformed 
channels; these movements represent specialized conditions. 
Many cells, especially egg-cells, exhibit streaming which 
is rhythmical but follows no preformed channels. This 
streaming can be observed in egg-cells particularly if they 
contain easily visible formed bodies or if having been 
bathed in a solution containing a non-toxic dye they took up 
particles whose movements can be easily followed. 

The best of the earliest descriptions of this type of move- 
ment in cytoplasm is that by Goette. 1 Since his time 
many workers, among them von Erlanger, 2 have studied 
the phenomenon. In living eggs one can of course fol- 
low these changes from minute to minute. So-called 
resting eggs — i.e., those that remain in a given stage, as for 
instance mature but unfertilized eggs — do not show this 
type of movement. If such mature resting eggs be fer- 
tilized, then the cytoplasm begins to stream. During each 
cleavage-cycle there is a period of little movement and a 
period of intense movement; these periods appear to parallel 
the stages in the cycle of nuclear changes. 

With the growth of the asters these movements become 
most marked. They begin over the spindle poles and move 
toward the spindle equator. As the opposing streams meet, 
they have only one possible direction: they can not move 
outward because of the resisting surface and thus move 
towards the centre of the egg. In this way the currents 
foreshadow the future cleavage plane; before the actual 
division of the cell-body the currents have brought about 
that disposition of inclusions found when the plane forms. 3 

Before fertilization the inclusions maintain constant posi- 
tions without reference to their specific gravity. If in such 
eggs they are segregated by means of centrifugal force, 

1 Goette, 1875. 

2 Von Erlanger, iSgj. 

3 Cf. Goette, iSjj, loc. cit. 



they return in time to their original positions. The oil is 
the last to return, I find, while the heavier yolk-spheres 
return first and after them the heaviest bodies, the pigment 
granules. After normal fertilization the inclusions shift 
positions and come to a new arrangement which again has 
no relation to their specific gravity. Thus, in the egg of 
Arbacia after fertilization the heaviest inclusions, pigment 
granules, remain close to the surface; the oil, shifting back 
and forth fairly even in distribution except as influenced by 
the mitotic spindle, forms clusters which mass and breakup 
again; 1 the yolk is evenly distributed outside the spindle 
area. The behavior of the pigment granules is due to the 
fact that they are trapped at the surface; they move only 
as the cleavage furrows are formed. In some eggs, as those 
of Nereis, the yolk and oil come to be massed and are defi- 
nitely distributed always to certain cells. These facts. lead 
us to assume that the flow in the cytoplasm is not every- 
where the same and that there are currents which are cir- 
cumscribed and limited to definite areas of the cell. If 
there were only one current of equal velocity in a medium 
everywhere the same, then we should expect a distribution 
of particles according to specific gravity, as is for instance 
the case in the blood-stream in a blood-vessel. 

The fact that cytoplasmic streaming in eggs is most 
expressed directly following the ectoplasmic changes, which 
result from the fertilization-reaction, gives rise to the sug- 
gestion that the ectoplasm by its activity sets up a condi- 
tion which increases the currents in the cytoplasm. That 
increased fluidity of the cytoplasm might be brought about 
by the escape of substances from the nucleus is a less likely 
assumption for the cause of the increase in streaming. It is 
true that streaming is not easily discerned in an egg like that 
of Nereis, fertilizable before maturation, until after break- 

1 Just, 1927a. 



down of the germinal vesicle; but before this break-down 
come the intense ectoplasmic changes incident to fertiliza- 
tion. In an egg like that of Chaetopterus after the germinal 
vesicle breaks down the spindle goes to the metaphase and 
comes to lie at the periphery before fertilization; although 
this translocation of the spindle indicates streaming in the 
cytoplasm, this soon diminishes; most intense streaming is 
resumed after fertilization. The cytoplasm of the unfer- 
tilized sea-urchin egg when ripe for fertilization is as fluid 
as any that I know, the germinal vesicle having broken 
down some time before in the ovary, but cytoplasmic 
streaming of measurable degree is demonstrable only after 
fertilization. Moreover, this cytoplasmic streaming is 
rhythmical: during cleavage the tides ebb and flow. And 
always ectoplasmic activity heralds their flood. It may 
very well be that in all cells ectoplasmic behavior as a 
response to changes in the environment initiates cytoplas- 
mic streaming. 

In eggs so far used in the study of the currents in cyto- 
plasm the two streaming movements from poles to equator 
by opposing each other seem to bring about cell-division. 
Theories concerning their cause have not been wanting, the 
chief of which is that the currents are due to surface-tension. 

The theory that the division of the cell-body is caused 
by changes in surface-tension is upheld by many investiga- 
tors. Some maintain that the cell divides because of 
increased surface-tension over the spindle-poles, whereas 
just as many workers are certain that the increase is at 
the equator. In many quarters what is considered the 
strongest proof that the division of the cell is due to changes 
in surface-tension comes not from observations and experi- 
ments on the cells themselves but from study of oil-drops 
suspended in water or solutions. It is a curious fact that 
here too the theorists are not agreed: some contend that an 
oil-drop divides because of increased surface-tension at the 



equator, whilst others insist that the increase is over the 
poles. Despite this lack of accord concerning the efficacy 
of the infallible principle of surface-tension in the division 
of oil-drops, to say nothing of the cells themselves, the 
surface-tension theory persists. 

Adore than once I have protested against the widespread 
misuse of surface-tension in biological explanations and 
theories. 1 Drops of oil in water or solution constitute a 
liquid-liquid system; a suspension of eggs does not. Whilst 
every oil-drop is a homogeneous liquid, each egg, far from 
being homogeneous, is itself a suspension of oil-drops, yolk- 
spheres, etc. — of materials of different chemical structure 
and physical make-up in cytoplasm, the living continuum, 
itself heterogeneous. The surface of an oil-drop is a film 
of molecular dimension chemically identical with the inte- 
rior of the drop; the measurable surface of an egg is thou- 
sands of times the width of the film of an oil drop and is a 
differentiated structure built by the egg during its develop- 
ment as a germ cell. The fact that one can induce stream- 
ing in oil-drops which closely resembles cytoplasmic 
streaming does not warrant the conclusion that since an oil- 
drop divides because of changes in surface-tension, division 
of the egg or other cells is likewise due to changes in surface- 
tension. Models used in biology to prove the cause of 
vital phenomena may be interesting but after all they are 
literally only models — imitations of the real thing and of 
little value for the analysis of the conditions in living 

Equally weak is the support for the surface-tension theory 
derived from experiments which demonstrate that during 
the cleavage-cycle, eggs of sea-urchins reveal a rhythm of 

1 Hercik {1934) entered a strong protest against the indiscriminate 
adducing of surface-tension for explaining biological processes. 
In his opinion the significance of surface-tension is often over- 



resistance and susceptibility to certain experimental agents. 
As I pointed out in the chapter, General Properties of 
the Ectoplasm, unfertilized eggs in hypotonic sea-water 
disintegrate more slowly than eggs which are exposed to 
the same degree of hypotonicity when after insemination 
break-down in the ectoplasm takes place. This period of 
ectoplasmic changes having passed over, the eggs are more 
resistant than unfertilized ones. According to many 
observers this resistance persists until just before or at 
first cleavage when the eggs become susceptible. It is 
held by some that this susceptibility is due to increased 
surface-tension over the spindle-poles. 

Now sea-urchins' eggs, as we have seen, are first spheres, 
then ellipsoids and finally, by cleavage, two spheres. Does 
the supposed change in surface-tension take place while the 
egg is still a sphere, when it becomes an ellipsoid or as it 
forms two spheres? Or does the theory disregard entirely 
that stage during which the egg elongates in the direction 
of the spindle-axis? The theory demands the most exact 
fixing of the moment in the cleavage-cycle when the egg 
reveals its maximum susceptibility in order to relate this 
to the act of cleavage. Observations made too far apart 
on a lot of eggs developing at the same tempo could easily 
miss the moment of maximum susceptibility. If, on the 
other hand, closely set observations be made on poor eggs 
developing at varying rates and therefore not reaching 
first cleavage at the same instant, the moment of maximum 
susceptibility would be erroneously fixed as coming in the 
stage or stages of the intact eggs present whilst the disin- 
tegrated ones would be in other stages. 1 

1 Degree of hypotonicity should be great enough to give sharp 
results; transfer of eggs should be uniform as to number of eggs y 
amount of dilute sea-water and amount of normal sea-zvater into 
which eggs are transferred. 



In the chapter, General Properties of the Ectoplasm, 
was mentioned the method of study of the susceptibility of 
eggs by putting them in solutions of dilute sea-water. 
In the experiments of R. S. Lillie 1 40 per cent, sea-water 
and 60 per cent, tap-water gave a solution that destroyed a 
percentage of the eggs at once, others later. In my experi- 
ments 2 on the rhythmical susceptibility of eggs of sea- 
urchins to hypotonic sea-water during a cleavage cycle, I 
modified this method. Instead of exposing the eggs to a 
solution made up of 40 parts sea-water and 60 parts tap- 
water, I used much more dilute solutions, mostly one made 
up of one part sea-water and 90 parts tap-water. On eggs of 
E chin arachnitis I have also used 10 cc. of tap-water to one 
drop of a sea-water suspension of eggs, an even greater 
dilution. The value of this method lies in the sharp 
results that it furnishes. 

I do not wish to imply that I discount the worth of the 40 
per cent, sea-water dilution as a means of revealing the 
susceptible periods. On the contrary; for I appreciate the 
fact that because it destroys eggs exposed to it during such 
periods, whilst it does not immediately arrest development 
of eggs exposed during their resistant periods, it is an excel- 
lent experimental means. I have used this dilution as well 
as others successfully; but I have found that its use should 
be checked by that of the greater dilutions mentioned 
above, for the reason that eggs exposed to lesser dur- 
ing terminal stages of resistance prior to the onset of the 
susceptible period might develop into this period. In 
such cases the eggs do not break down in the stage which 
they had reached at the moment of exposure. The dilu- 
tions therefore should be so great that they halt develop- 
ment abruptly; then the time in seconds from the moment 

1 Lillie, R. S. ? 1916. 

2 Ju*U J922e; ip2Sd. 



of exposure to disintegration can be attributed wholly to 
the dilution's destructive action and not in part to a pos- 
sible anesthetic action. 

The use of extremely dilute solutions was not my only 
modification of method. I exposed the eggs during a 
cleavage-cycle at the short intervals of one and two minutes. 
Thus, I was able, using always eggs developing at the same 
tempo, to define with sharpest exactness the briefly endur- 
ing periods of susceptibility which otherwise would have 
escaped observation. 

I think that there can be little doubt that my experi- 
ments did more than confirm earlier ones which proved the 
existence of periods of resistance and susceptibility during 
the cleavage-cycle of the sea-urchin egg; they demon- 
strate exactly the onset and duration of the period of 
susceptibility. The significance of my findings for a 
theory of cell-division needs no elaboration. If we wish 
to base a theory of the cause of cleavage upon the occur- 
rence of rhythmical resistance and susceptibility parallel 
to the rhythm of cell-division, we first of all need accu- 
rately to relate the susceptible period to a definite stage in 
the process of cell-division. One finely spun physico- 
chemical theory of cell-division falls to the ground because 
its author failed to time precisely and to relate definitely 
the onset and duration of that period of susceptibility 
which precedes the appearance of the cleavage-furrow. 1 

Perhaps the simplest way to begin the discussion of my 
findings is to present the data of a typical experiment, on 
the egg of Arbacia punctulata.- Accordingly, these data 
in the form of a table (Table III) are herewith given. The 
reader will note: First, there are two periods of susceptibility 

1 Lillie, R. S., 1916. 

2 The egg of Echinarachnius which is less resistant than that of 
Arbacia reveals much more sharply the periods of susceptibility. 



during the cleavage-cycle of the egg. It is the second, the 
greater, which comes prior to the appearance of the cleav- 
age-furrow, with which we are primarily concerned. 
Secondly, during both periods the egg is spherical. The 
change in form from spherical to ellipsoid occurs once only 
during a cleavage-cycle; it marks the end of the period of 
greater susceptibility. These two findings possess especial 
significance for an interpretation. 

Table III. — The Rate at Which Eggs of Arbacia Disintegrate in Distilled 
Water at Intervals after Insemination* 

Time, in 

Time, in 



seconds, to 




















18. s 






23 -5 


Streak indicated 


























"Dumb-bell stage" 














Eggs burst over spindle pole 




Eggs elongate 




Cleavage furrow indicated 





* Eggs of Arbacia are inseminated, and at intervals after insemination 5 drops of them are mounted 
under the microscope in 15 cc. of distilled water. The first column gives the number; the second, 
the time after insemination; the third, the time to cytolysis; and the fourth, the stage of the egg at 
the time of exposure. 



As regards the first named finding, that there are two 
periods of susceptibility in a cleavage-cycle, I should 
like to point out that an interpretation based on the 
occurrence of the second which overlooks the first is diffi- 
cult to defend. If susceptibility as such is the cause for 
cleavage, then the egg should cleave after each such period. 
This is not the case. Further, it must be remembered that 
in many eggs the most pronounced period of susceptibility 
occurs within a minute after fertilization — that is, depend- 
ing upon the species of egg, anywhere from 34 s or M20 of 
the total time to first cleavage, at a time long before the 
onset of the first cleavage-cycle which, as stated above, 
begins only with the stage of apposition of the egg- and 
sperm-nuclei. Also if we attempt to correlate the sus- 
ceptibility with nuclear phenomena, we find ourselves at a 
loss. During this period of susceptibility immediately fol- 
lowing fertilization, neither the sperm-nucleus nor the egg- 
nucleus is in active mitotic condition. In that period of 
susceptibility which occurs first in a cleavage-cycle, the 
apposed nuclei are not in mitosis. Thus only in that sus- 
ceptible period immediately prior to cleavage is there 
present an active mitosis. Then susceptibility does not 
depend upon or run with a definite stage in mitosis. It 
follows from this that all the learned disquisitions relating 
the origin of the period of susceptibility to the nuclear 
state and more specifically to the behavior of the asters 
have no basis in fact. 

Whilst it is true that the periods of susceptibility can not 
be correlated with mitotic phenomena, they can be with 
ectoplasmic state or behavior. At the first period of sus- 
ceptibility that appears during a cleavage-cycle, the ecto- 
plasm manifests peculiar behavior as it does during the 
period of susceptibility immediately prior to the act of 
cleavage. The greatest observed susceptibility is that 
occurring immediately after fertilization; in this stage the 



egg exhibits the strongest ectoplasmic changes ever shown 
in its development. The periods of susceptibility thus 
can be related to structural and behavior-changes in the 
ectoplasm. No other constant change being associated 
with the period of susceptibility than the visible and easily 
demonstrable ectoplasmic state, we can assume that 
ectoplasmic state is wholly or in part responsible for the 
temporary lowered resistance of the egg to dilute sea-water. 

Not only did I fix the period of maximum susceptibility 
of sea-urchins' eggs during their cleavage-cycle to hypo- 
tonic sea-water; I was able also to localize precisely the 
point on the eggs at which they break down during this 
period. Properly to appreciate this latter finding, we need 
fully to understand the structure of the hyaline plasma- 
layer. This, part of the ectoplasm, though often the subject 
of discourse by many writers, has never been properly 
understood. I give therefore a detailed description of the 
origin and structure of the hyaline plasma-layer in sea- 
urchins' eggs, thus supplementing the description given in 
the chapter on the ectoplasm. 

Selenka 1 years ago described the surface of various echi- 
noderm eggs as composed of a sheath of clear cytoplasm. 
Later Hammar 2 and others also described this external 
layer on fertilized sea-urchins' eggs. It is variously known 
as the ectoplasmic layer, hyaline plasma-layer, Hammar's 
layer, etc. It should not be, as it often is, confused with 
the vitelline membrane. 3 

I have found that in several species of sea-urchins this 
hyaline plasma-layer arises in the same way and has much 
the same structure as described for the fertilized egg of 
Arbacia. Into the perivitelline space which arises with sep- 
aration of the vitelline membrane fine filaments of the cvto- 

1 Selenka, 1S7S, 1SS3. 

2 Hammar, 1S96, 1S97. 

3 Just, I930g, 19330. 



plasm project. 1 By anastomosis of their free ends a very 

fine covering membrane forms. 2 Thus the hyaline plasma- 
layer is clear granule-free cytoplasm in the form of finely 
spun threads covered by a thin membrane. Other eggs, as 
shown in the chapter, The Ectoplasm, possess these ecto- 
plasmic filaments. They are present on every marine egg 
that I know. Unfertilized sea-urchins' eggs treated with 
strong hypertonic sea-water also show that the surface- 
cytoplasm is made up of radial strands. Eggs of Nereis 
and of Platynereis show especially well after fertilization 
immediately below the vitelline membrane a delicate 
plasma-membrane, which is the covering film of cytoplas- 
mic prolongations. It is these prolongations, of greater 
length and diameter than those of sea-urchins' eggs, which 
give the surface of the eggs of these worms their striated 
appearance. These prolongations constitute the outer 
region of the ectoplasm. They and their covering film 
compose the hyaline plasma-layer of the egg. 

Others before me have described on the fertilized egg 
in later stages this layer as made up of filaments. Meves, 3 
for example, has pointed out that some time after fertiliza- 
tion the layer is composed of threads and a thin covering 
membrane. This structure of the hyaline plasma-layer 
can be demonstrated by placing fertilized eggs in calcium- 
free sea-water; the covering membrane becomes destroyed 
leaving the filaments free. 

During the whole cleavage-period, sea-urchins' eggs 
show on the surface of each blastomere these fine cyto- 
plasmic projections covered by a delicate membrane which 
together make a continuous film enclosing the blastomeres. 
The filaments are most easily seen between adjacent blasto- 
meres as they are separating at cleavage. Since there is no 

1 Cf. also Berthold, 18S6, and Theel, 1S92. 

2 See also Mrs. Andrews. 

3 Meves, igi2. 



great decrease in width of the hyaline plasma-layer as cleav- 
age progresses and since by treatment with calcium-free 
sea-water the presence of the filaments can be demonstrated 
on eggs throughout cleavage, we reach the conclusion that 
the egg builds new hyaline plasma-layers constantly. One 
can not say exactly where in the cytoplasm the hyaline 
plasma-layer begins, for each filament, the essential struc- 
ture of the layer, is a granule-free prolongation of the 
cytoplasm. The activity — amoeboid, spinning, and con- 
tractile—of the hyaline plasma-layer, so well described by 
Mrs. Andrews as early as 1897, is due to the behavior of 
these continuations of the egg-plasma. The filaments of 
the hyaline plasma-layer are a living part of the living pro- 
toplasmic system (see also Fig. 36). x 

As a living and integral part of the egg, these filaments, 
we should expect, react as the cytoplasm of which they are 
continuations. Because of their tenuous and granule-free 
structure they are physiologically different from the remain- 
der of the egg. However, any very striking difference in 
their physical behavior needs to be fully established. It 
was claimed by Goldschmidt and Popoff 2 first and later by 
Gray 3 that hypertonic sea-water increases the Volume of 
the hyaline plasma-layer and decreases that of the egg and 
thus reveals a marked difference between the osmotic 
properties of the surface-cytoplasm and the remainder of 
the egg. If this interpretation were correct, I would call 
this difference in osmotic properties — what Gray calls a 
fortunate coincidence — a most important discovery. We 
should use every available fact concerning the behavior of 
colloids in living protoplasm. Is it true that the hyaline 
plasma-layer increases in volume whilst the remainder of 

1 Ectoplasmic protrusions on living dividing cells have been early 
described by many zvorkers. 

-Goldschmidt and Popoff \ 190S. 
z Gray, 1931. 




the egg decreases because "the former is freely permeable 
to electrolytes whereas the latter is not" ? x The answer is 
simple: the existence of such a difference has actually not 
been proved. 

The correct interpretation of the changes in sea-urchins' 
eggs induced by means of sea-water made hypertonic by the 
addition of either electrolytes or non-electrolytes is as fol- 
lows: placed in hypertonic sea-water, the whole egg, includ- 
ing its ectoplasmic filaments, loses water and shrinks. The 

Fig. 36.— Cleavage of an egg of Echinus microtuberculatus in calcium-free 
sea-water (after Herbst). The covering membrane of the ectoplasm is destroyed, 
leaving the filaments free. 

filaments decrease in diameter and appear as lines thereby 
becoming more easily visible. In every egg known to me, 
the ectoplasmic filaments appear in the same way as an 
effect of hypertonicity. 2 

We can not escape these facts: that after fertilization and 
with membrane separation the surface of the egg is studded 
with filaments; and that also unfertilized eggs as they shrink 
in hypertonic sea-water show these filaments. It is patent, 
therefore, that the filaments are not new formations called 
forth by the action of electrolytes. The action of electro- 
lytes does not demonstrate a difference in permeability 
between endoplasm and ectoplasm. 

Gray also errs when he says that the hyaline plasma-layer 
dissolves in calcium-free sea-water. Rather, eggs in this 

1 Gray, Lc. 

" Cf. Faure-Fremiet. 



medium show the filaments very beautifully as the figure 
taken from Herbst so clearly shows 1 (Fig. 36). Herbst 
himself thought that he had altered the structure of the 
hyaline plasma-layer, changing its homogeneous structure 
to a collection of radial threads whereas actually by the 
treatment with calcium-free sea-water, he only removed 
the covering membrane of the threads. This change in 
the eggs' environment prevents the formation of a new 
membrane which takes place when the eggs are returned to 
normal sea-water. The so-called removal of the hyaline 
plasma-layer by microdissection is also a removal only of 
the covering membrane. 

Goldschmidt and Popoff advanced the idea that the ecto- 
plasmic filaments on the sea-urchins' egg are due to astral 
radiations from the nucleus. But there are no astral radia- 
tions in an unfertilized egg momentarily subjected to the 
action of strong hypertonic sea-water; nor are any present 
in an egg fifty seconds after insemination. In both cases, 
filaments can be observed. Fertilized sea-urchins' eggs 
shrink slightly when the astral rays are first most extensive 
so that the eggs 5 surface appears somewhat crenated because 
the filaments stand out strongly. It is this crenation that 
these authors observed. 

Gray and Goldschmidt and Popoff have derived theories 
of cell-division from their observations on the behavior of 
the hyaline plasma-layer. Since these observations are 
incorrect, the interpretation given them is unwarranted. 
This is thus another example of the danger of applying 
physico-chemical notions to a biological process in the 
absence of knowledge of the underlying structural condition. 

During a cleavage-cycle of sea-urchins' eggs the ecto- 
plasm undergoes changes. Until the stage 2 when the 

1 Herbst, iSgg. 

2 See Ziegler, 1904. Compare Ziegler, /SgS, on egg of Beroe. 



chromosomes are at the anaphase and the egg is still spheri- 
cal, the hyaline plasma-layer is everywhere of equal width 
and the filaments in it are all the same length. In later 
anaphase or early telophase the ectoplasm shows a rapid 
change, moving from over the spindle poles toward the 
equator, the site of the future cleavage-plane, in an amoe- 
boid or wave-like fashion. The ectoplasm then begins to 
move inward toward the egg-centre, in the direction of the 
course of the cytoplasmic currents, and the egg elongates. 
As the ectoplasm continues to move inward, the covering 
film of the hyaline plasma-layer resists. Thus the moving 
ectoplasm exerts a pull on its filaments in the region of the 
oncoming cleavage plane so that they are longer here than 
elsewhere. With the beginning of the moving of the wave 
of amoeboid changes from the area over the spindle poles 
to the equator, the filaments in the former region appear 
to lose connections with their covering membrane, whilst 
those in the latter seem more firmly bound to it. 

By the very simple method of treatment with extremely 
dilute sea-water, I was able to ascertain that while the 
egg is still spherical and when the ectoplasm exhibits 
the wave-like movement from the circumpolar areas to the 
equator, it has definitely localized weaknesses in this, the 
most susceptible, period of its cleavage-cycle. At this time 
the egg breaks down in about ten seconds after immersion 
in the dilute sea-water by an outflow from the areas over 
the spindle-poles. Though eggs of some species of sea- 
urchins show this localized disruption more clearly than 
others, it can be observed in all; none ever show an exuda- 
tion elsewhere than from the circumpolar areas. With 
elongation the egg is again resistant. 

As we have seen, the eggs 5 period of greatest susceptibility 
to hypotonic sea-water occurs during the process of mem- 
brane-separation. At this time the ectoplasmic filaments 
appear more sharply because of break-down of material 



at the surface of the egg. Though this susceptibility is 
not of immediate concern here, since it does not relate to 
the cleavage-cycle, we adduce it since it shows susceptibility 
as due to ectoplasmic change. In the first period of sus- 
ceptibility which occurs during the cleavage-cycle and 
which falls in with the stage of union of the egg- and sperm- 
nuclei, when there is a normal shrinkage of the egg-contents 
and the ectoplasmic filaments are most easily visible, the 
egg does not disrupt at definite points. In some other 
eggs, those of Crepidala, 1 for example, it seems that the 
weakness that appears at this time or soon after, persists. 
In the period prior to cleavage, there are localized points 
of weakness due to ectoplasmic activity; they are limited to 
the egg-poles. In all periods, susceptibility is due to 
changes in the ectoplasm and where these are local, break- 
down of the egg is likewise localized. 

In none of these three periods of susceptibility can the 
break-down of the egg be ascribed to changes in surface- 
tension. It is not possible to regard the complex structure 
of the ectoplasm with its palisade arrangements of filaments 
and their covering membrane as a simple surface-film. 
The width of this covering membrane alone surpasses by 
far that of a molecular film. Besides, this membrane is 
passive in cell-division; ectoplasm and filaments play the 
active role, changing from moment to moment first in one 
region and then in another. These changes in structure 
and behavior parallel the periods of susceptibility. 

The ectoplasm of marine eggs is a highly mobile, con- 
stantly changing structure. In eggs as in other cells, 
protozoan, nerve, muscle, etc., it is no mere inert sheath. 
Instead, it reflects in its spinning, in its suceptibility, the 
irritability of the living system in response to environmental 
changes. In addition to the significance that its general 

Conklin, 1912b. 



properties have for cell-life, the ectoplasm by its behavior 
as here described in the cleavage-cycle, plays a definite 
role in the process by which the cytoplasmic mass becomes 
two cells. 

When a cell divides, the plane of cleavage arises either 
from within the cell-mass and extends outward, or from the 
periphery and extends inward. The former mode, cleavage 
by formation of the so-called cell-plate, is frequently met 
with in plant cells. Division of animal cells takes place by 
a furrow extending from the periphery inward. Cleavage 
by formation of a cell-plate in animal cells is only seldom 
found and is a modified process occurring together with 
furrowing. The problem of cell-division for animal cells is 
that of the origin of the furrow which separates the dividing 
cells. The phenomena observed during the process of fur- 
rowing in the living sea-urchin eggs by which the single 
cell becomes two we summarize as follows: 

Currents in the cytoplasm move toward the site of the 
future cleavage-plane; meeting there the opposing streams 
they move inward. The ectoplasm, in whose behavior lies 
the cause of streaming, then actively shifts by amoeboid 
movement from the circumpolar areas toward the equator. 
With this the egg quickly elongates. The ectoplasm moves 
then inward along the plane pre-delineated by the cyto- 
plasmic currents. Cleavage in a sea-urchins 7 egg is there- 
fore accomplished by active movement of the ectoplasm. 

Neither cytoplasmic movements nor changes in ecto- 
plasmic behavior are confined to animal eggs. Cytoplasmic 
streaming has been described in plant cells, especially by 
Ewart. Movement in the cytoplasm of Protozoa is well 
known. Changes in shape and in activity of the ectoplasm 
are similarly familiar from descriptions made by the earlier 
workers on cells. Recently, investigators using the method 
of tissue-culture have, particularly by means of the cine- 
matograph, recorded the evanescent changes at the cell- 

2$ 4 


surface which accompany cell-division. They are also 
well defined in other living cells when freshly dissected out 
of the organism. We need not, therefore, confine our 
explanation of cell-division to eggs of sea-urchins. We 
dismiss as cause for cell-division division of the nucleus, by 
mitosis with or without asters, and by amitosis, whether 
occurring simultaneously with cytoplasmic division or not, 
since no constant relation between nuclear division and that 
of the cell-body can be established. Since not all cells are 
spherical and not all spherical ones elongate during the 
cycle, we dismiss also elongation of the cell as a factor. 
Movement in the cytoplasm depends upon ectoplasmic 
activity; it is thus not a primary factor. There remains 
the ectoplasm found in all cells. 

Cells do not extend indefinitely into space. They possess 
surfaces. Cell-division means that one cell becomes two 
by the rise of a new partition. Does this arise within the 
cytoplasm or does it come about through the extension of 
the ectoplasm inward? Until we know more concerning 
the origin of cleavage by cell-plates, we may only hazard 
conjectures as to the formation of cell-walls within the 
cytoplasm as found in plants. For animal cells, generally, 
we must seek the cause of cell-division in ectoplasmic 



I I 
Cleavage and Dijfere?itiatio?i 

The essential problem of animal development lies 
in the question; How does the egg, a single cell, become an 
adult organism? If one take the transparent eggs of a 
common marine fish like the mackerel which float like 
bubbles on the surface of the sea, one can easily follow their 
development under low power of the microscope. Before 
fertilization a thin film of cytoplasm beneath the membrane 
encloses a core of yolk and oil. With fertilization this 
cytoplasm flows to one egg-pole to collect there as a disc. 1 
This disc is next crossed by one furrow and then by another 
at right angles to it: so by this cleavage two and later four 
cells arise. Cleavage progresses until many cells form, 
whilst some of the yolk beneath the cytoplasmic disc is 
transformed into cytoplasm. Soon one discerns an opaque 
line running the length of the disc. Here there are more 
cells than elsewhere, hence the opacity; from some of these 
will come the future embryo. Under the microscope even 
one who has very little knowledge of the development of 
eggs can follow the origin and formation of the nerve tube 
out of which the brain and spinal cord emerge. One looks, 
as through an open window, at the very mystery of life, 
wondering at the heartbeat, first uncertain and then in 
definite, sure rhythm; the bright red blood moving in jerks 
with each beat of the heart; the first spasmodic muscular 
twitch; the appearance of the purple-black eye-pigment; 
the definite fish form; the color of the skin which will give 

1 Cf. Ransom, fSj)j. 



the markings that make the adult mackerel one of the most 
beautiful creatures of the sea. 

There are other eggs, those of ascidians, which like the 
mackerel belong to the highest group in the animal king- 
dom, that hatch as swimming larvae in eight hours after 
fertilization instead of seventy-two hours, the time the 
mackerel egg in a warm laboratory requires. From egg to 
complex animal in eight hours ! The problem of the devel- 
opment from a single cell to a complex animal is thus a 
fascinating one. 

In the following exposition we shall see again that exact 
description of the process which we aim to explain plays 
a chief and significant role. Whilst here as with many 
other problems in biology we must realize that a description 
does not explain the process described, nevertheless descrip- 
tion is the prerequisite of a successful attack on the 
problem of development, a problem which doubtless more 
than any other in biology is complicated by the interplay 
of simultaneously occurring reactions and by rapidly suc- 
ceeding events. To determine cause and effect in this 
manifold process of differentiation is a task whose difficulty 
is so manifest that we must before all else seek to follow 
the process as exactly as possible in closely set stages. 
We may then be able to relate smallest changes in form and 
in expression to general phenomena of development, that 
is, to the differentiation which accompanies the cleavage- 
process. Such a task is undertaken in this chapter. 

Following the classification of Karl Ernst von Baer, one 
can separate the development of fertilized or parthenogenetic 
animal eggs into four periods: cleavage, formation of the 
germ-layers, development of the organs and histological 
differentiation. This classification represents a scheme 
which holds more nearly true for the higher animals than 
for the lower and should not be taken as meaning that the 
development of any egg is sharply divisible into fixed 



periods. Development is a catenary process of overlapping 
stages and can not be categorically separated by rigid lines. 
With periods three and four, which have to do with the 
establishment of the organs and the finer differentiation 
within them, we have here no concern. Period two includes 
the formation of the primary sheets of cells, the ectoderm 
and the endoderm in the lowest multicellular animals, the 
sponges and the coelenterates, and the ectoderm, endoderm 
and mesoderm in all the other multicellular animals. 
Period two represents the end-result of period one. It is 
in this first period, cleavage, that our problem lies. And 
this for the following reasons: 

Coming first, this differentiation during cleavage stands 
nearest to the condition in the egg at the moment of fer- 
tilization, a moment that forms in time the limit between 
the condition of the egg as single cell and those conditions 
of the egg as multiplying cells which we call the differentia- 
tion during cleavage. Hence studying the period of cleav- 
age, we approach the source whence emerge the progres- 
sively branched streams of differentiation that end finally 
in almost quiet pools, the individual cells of the complex 
adult organism. 

But just as the source of a river though single may not 
be simple but compounded of rivulets, so the fertilized egg 
though single is not simple, being itself a complex of many 
contributary streams of differentiation. For the fertilized 
egg is already a differentiated system. Indeed, the whole 
history of the egg from the moment when it became dis- 
tinguished as such and separated from body cells is a suc- 
cession of differentiations. In form, growth, mode of 
building up of food reserves, and in nuclear structure, a 
young egg-cell shows itself different from every and any 
other cell of the animal's body. The egg-cell is subjected 
to the same environmental influences as other, fully special- 
ized cells of the animal's body. The changes and processes 



by which it becomes an embryo thus spring from an initial 
differentiation, an intrinsic organization of the egg that 
distinguishes it from all other cells. 

Consider the chick at the moment when it emerges from 
the egg-shell: it has all the organs that it will ever have 
and it had the precursors of these at the moment that it 
was fertilized. Nothing of organic material did it gain 
during the 21 days of incubation. It took in water, oxygen 
and heat. But not one single milligram of living proto- 
plasm is in the hatched animal but what was within the 
unincubated egg. Early the embryo formed blood with 
a red pigment; this pigment contains iron. But not the 
most minute fraction of this iron was gained during devel- 
opment; all present in its body at hatching was already 
there before the egg was laid. 

True, a mammalian embryo, like that of the human, 
develops by the material of the blood which comes to it. 
But in the minute human egg must be present all that from 
which comes a human embryo for the blood which nourishes 
it is the same as that which supplies any other cell in the 
mother's body, no one of which becomes a human embryo. 
Hence, though the human egg differs from the bird's, at 
basis the same is true: the egg at beginning of development 
is a differentiated and a differentiation-capable system. 

Following its period of primary differentiation the egg 
cell grows, reaching a stage prepared for fertilization but 
unfertilizable. Then it becomes fertilizable — another phase 
of differentiation. If fertilization ensues, it becomes dif- 
ferentiated again. Thus differentiation flows on as a stream 
without breaks and if later for purposes of convenience I 
use words that seem to connote sharply discontinuous 
phases, the reader should keep in mind that I regard these 
phases, periods or stages, only as moments in time when 
the stream alters its course. Although we can not yet 
assert what in all this history is cause and what effect, 



nevertheless we may say in general terms that each suc- 
ceeding differentiation is conditioned in part by extrinsic 
and in part by intrinsic factors, the latter of which are 
conditioned by the preceding differentiation. 1 One great 
task in the study of the differentiation occurring during 
cleavage is the analysis of these factors, a task especially 
difficult because we have no known point from which we 
may start, for in this respect the egg also must be regarded 
as an unknown system. 

In the second place we concentrate attention on the 
cleavage-period in our effort to trace the differentiation 
of development, because cleavage is a process common to 
all animal eggs. Every species of animal egg passes through 
cleavage, but not every animal egg, e.g., of sponges and 
of coelenterates, passes through the three other periods 
enumerated above. Then the differentiation that takes 
place during cleavage we regard as that embracing in 
a wider sense those characteristics common to animal 
development and most readily reducible to general terms. 
As we shall later see, whatever the period of development, 
in which differentiation reveals itself to us, the mode of 
differentiation is always the same. This being true, study 
of the differentiation in the most generally appearing stage 
has distinct advantage. 

In the third place we have the practical reason that, as 
experience has taught, of all the periods of development the 
cleavage period lends itself most readily to resolution of 
the processes into closely set stages; thus by experiment it 
is possible to analyze the factors which set up the conditions 
for differentiations in a more normal or natural manner 
than, for example, in experiments with transplantations 
involving conceptions of "organizators" and the like. 

1 Cf. Delage, 1S95, pp. 765-766. 



For these three capital reasons, then, I consider it best 
to limit my discussion of differentiation to the cleavage- 
period. Since the fertilized uncleaved egg is itself dif- 
ferentiated, and in this respect an unknown system, a 
distinct advantage accrues in pushing back our inquiry as 
far as possible to it as a single cell whence originate the many 
different kinds of cells that make up the embryo. For the 
great question is: how out of a single cell do these manifold 
differences arise? I repeat: singleness does not mean sim- 
plicity. And yet, inasmuch as the complex organism has 
its genesis in a single protoplasmic system, the unknown 
complexity of this system may in some measure become 
known by means of a resolution of the events by which a 
single cell organism becomes one of many cells. It follows 
from this that we should define very exactly the cleavage- 
period. I turn therefore to a brief resume of cleavage 
processes in various animal eggs. Such a resume will 
further be valuable, because in the later discussions I 
shall need to refer to the various cleavage-types; and 
because by evaluating differences in the mode of cleavage 
Ave may arrive at a basis of similarity. 

Every developing animal egg normally passes through a 
period of successive cell-divisions or cleavages. The 
cleavage-cells or blastomeres always arise by binary fis- 
sion, that is, two " daughter-cells " arise from the division 
of one cell. In this fashion is established a cleavage-pattern 
which differs with different eggs. Upon these differences 
depends the classification of eggs according to their type of 
cleavage. Cleavages are classified as total and partial: 
total, if the whole egg cleaves; partial, if only a part of the 
egg cleaves — this cleaving portion is always either the whole 
surface or a definite surface-area. 

In total cleavage the blastomeres of first cleavage and 
of several subsequent divisions may be approximately equal 



in size; they may be at first equal in size and early in the 
succeeding cleavages show marked size-differences or they 
may from the first cleavage show striking disparity in size. 
At the end of the cleavage-period the blastomeres are of 
unequal size in all totally cleaving eggs. 

The position of the blastomeres also plays a part in 
making the cleavage-pattern in totally cleaving eggs. If 
they stand directly above each other the final pattern is 
radial; if the four smaller cells, instead of lying directly 
over the larger ones, lie above the furrows of the first and 
second cleavage, the pattern is spiral. 

The derivation of the blastomeres may be so regular 
that one can determine exactly in the cleavage stages the 
time and place of the origin of each. For many eggs it 
has been shown that the blastomeres in late cleavage can 
be traced unerringly back to the stage of first division. 
Thus, for these eggs, a cell-lineage, as it is called, can be 
traced. In other eggs for several cleavages the blasto- 
meres arise always in the same way and hence show con- 
stancy in origin; after this period, they vary both as to 
time and place of origin. In other words, the cleavage 
pattern of animal eggs may be constant or not. If their 
cell-lineage can be traced, the pattern so far as the origin 
of the blastomeres is concerned, is constant. It is to be 
noted that the cleavage of eggs of clams, worms, ascidians, 
showing cell-lineage is closely similar. Hence this type of 
cleavage is not restricted to any one group of animal eggs. 
If the lineage of the cells can not be traced, this means that 
the cleavage-pattern is composed of cells whose positions 
are not determined by fixed origin. 

Partial cleavage is of two forms, discoidal and superficial. 
In the former, cleavage is confined to a disc at only one pole 
of the egg, whilst in the latter the entire superficial cyto- 
plasm cleaves. Discoidal cleavage is found in eggs of 
cuttle-fishes and their allies, in many bony fishes and in 



reptiles and birds ; superficial cleavage in eggs of some 
coelenterates, of many insects and of some other arthropods. 
Superficial cleavage might be said to be the mode among 
arthropod eggs for with few exceptions in the eggs of the 
members of this group that embraces the crabs, spiders, 
insects etc., only the surface-located cytoplasm shows 

In figures of the discoidal cleavage in the egg of an ink-fish 
or squid, Loligo^ as seen in section, one notes at the upper 
pole of the egg a heavily drawn line which represents the thin 
sheet of superficial cytoplasm. It is in this sheet that the 
cleavages ensue. In the unfertilized egg of a fish a thin 
band of cytoplasm encloses the yolk. After fertilization 
the superficial cytoplasm moves to the upper pole of the 
egg to form a disc where cleavage now takes place. In 
eggs of reptiles and of birds the process is the same: cleavage 
is limited to a small area of the total egg. As this disc 
becomes cleaved, the underlying yolk is converted into 
active cytoplasm which by cleaving adds to the area of 
the original disc. 

Superficial cleavage was first clearly described by Weis- 
mann in 1864. Since the appearance of this classic memoir 
on the development of the insect egg, the clear superficial 
cytoplasm of the egg, which encloses the egg-yolk and to 
which during cleavage cell-boundaries are confined, has 
been spoken of as the blastema. Following entrance of the 
spermatozoon into the egg through a canal, the micropyle, 
located at one pole of the ellipsoid egg, the egg- and sperm- 
nuclei come together and form the cleavage nucleus located 
below the egg surface. This nucleus divides mitotically sev- 
eral times without cleavage of the egg. Later most of these 
daughter nuclei become located in the blastema, a few 
remaining in the central yolk-mass. With the arrival of 
the nuclei in the blastema, cleavage planes appear in it, 
the interior of the egg remaining uncleaved. 



The foregoing general statement on the cleavage-process 
in animal eggs ; though it makes no pretension to be exhaus- 
tive, nevertheless covers the essential points on a process 
which has been the subject of much admirable and pains- 
taking investigation. In a book like this a comprehensive 
treatment of cleavage would be out of place, because it 
demands more space than we can give it. Moreover, a 
certain advantage obtains here, as with other biological 
processes, in setting forth a plain and simple statement of 
salient features that stand as accepted and established facts. 
Without further discussion we may note the following 
points concerning the cleavage-process in animal eggs: 

1. Cleavage may involve the whole or only a part of the 
animal egg. Hence, the pattern varies depending upon 
the area of the egg which undergoes cleavage; the pattern 
at the termination of the cleavage-period resulting from 
total cleavage, as in the egg of a snail, is markedly different 
from that of its near relative, the squid, which undergoes 
partial (discoidal) cleavage. 

2. The size of the blastomeres contributes a distinguish- 
ing feature to the cleavage pattern. Where in developing 
insect eggs the cell-size has been investigated, it was found 
that the blastomeres in the superficial cytoplasm show 
equality in size. This is not true for the cleavage-cells in 
the eggs of the ink-fish and in other eggs exhibiting discoidal 
cleavage. In total cleavage the blastomeres may be mark- 
edly unequal in size. Hence, not only the extent of 
the egg undergoing cleavage, but also size-differences of 
the blastomeres in the cleavage-area constitute a factor 
which determines an egg's cleavage pattern. 

3. In all forms of cleavage, except superficial, the initial 
splitting up of the egg-substance takes place at that pole 
from which the polar bodies have formed or will form. In 
total cleavage the first and second cleavage planes cut 
through this pole at or nearly at right angles to each other. 



In discoidal cleavage the cleaving disc is located at this 
egg pole. In superficial cleavage, the initial nuclear divi- 
sions are confined to the region directly below the point 
whence the polar bodies form, whilst the cleavage itself 
involves the whole egg-surface. 

Because hitherto some writers have preferred to assume 
a simplicity for the uncleaved egg, thus disregarding its 
previous history as a succession of differentiations, fruit- 
less attempts have been made to interpret the cleavage- 
patterns of the animal egg, diverse though they are, as the 
mode for or even the cause of the setting-up of those dif- 
ferences whose sum-total defines the differentiation made 
manifest during cleavage. It is, however, more cor- 
rect to see in the cleavage-pattern a display of the differ- 
entiations present before cleavage set in. The separa- 
tion of the egg as egg from other cells of the organism, 
its position in the ovary, its relation to food-supply, its 
intake and elaboration of food both in quantity and quality 
— in short all those processes that long before fertiliza- 
tion and cleavage endowed the eggs with those character- 
istics to which many authors relate cleavage as total or 
partial and as variants of each of these — these are the proc- 
esses which condition the respective cleavage-patterns. 
Thus, a cleavage-pattern is established by earlier occurring 
differentiations; it does not express differentiation occurring 
during cleavage. We therefore consider the cleavage- 
pattern as only the frame-work within which we try to 
find the answer to our question. By analysis of the events 
which occur during cleavage, we may reach an agreement 
upon a proposition as to the primary and basic factor which 
underlies all forms of differentiation. For the cleavage 
these chief events are six. 

(i) The egg is split up into cells; (2) the embryonic axis 
and the plane of bilateral symmetry are revealed; (3) the 
cytoplasmic inclusions shift positions; (4) nuclear material 



increases; (5) water is redistributed; (6) the ectoplasm 

Before I begin the discussion of these events, I must deal 
with the loss by an egg of capacity to produce more than 
one embryo — i.e., with the change of the egg from a pluri- 
potent to a unipotent system. This loss of pluripotency, 
often spoken of as a chief problem in development, in my 
judgment, is only a revelation that embryogenesis is a series 
of progressive restriction. 

Loss of pluripotency is revealed at that stage in cleavage 
when the blastomeres having been experimentally separated 
develop into defective embryos. In the eggs of snails, 
for example, this loss can be demonstrated to have taken 
place at first cleavage; blastomeres separated at this time 
develop each to a swimming form, which is an incomplete 
embryo made up of those structures which in the normal 
embryo would have arisen from this blastomere. In eggs 
of echinids, on the other hand, loss of pluripotency occurs 
later because only after the third cleavage do the blasto- 
meres when separated develop into defective embryos; 
blastomeres isolated after first and second cleavage develop 
into perfect though dwarf embryos, one-half and one-fourth, 
respectively, the size of the normal. Briefly, we find that 
animal eggs vary with respect to the time after fertilization 
when they lose capacity for multiple embryo-production. 

Cleavage has been classified as determinate and inde- 
terminate. In eggs with determinate cleavage, the history 
of each cell or blastomere in a late period of the cleavage- 
process can be traced back to the two cell stage; that is, 
some eggs as early as the two cell stage reveal an organiza- 
tion which fixes the destiny of the blastomeres derived 
from the products of the first cleavage. In so-called inde- 
terminate cleavage, although for a time the blastomeres 
show constant origin, they later arise in undetermined 
fashion. This classification of the cleavage of eggs as 



determinate and indeterminate I omit from the discussion 
for two reasons. First, since in an indeterminate egg, as 
that of an echinid, the initial cleavages are determinate, 
it can not be called strictly indeterminate. Second, since, 
all monoembryonic eggs finally become unipotent, we can 
not say that only determinate eggs are unipotent. 

The evidence at hand shows clearly that in one respect 
eggs are the same: I refer to the fact, never sufficiently 
emphasized, that before fertilization all eggs, amenable to 
the experiment of merogony, have capacity to produce 
many embryos. 1 If eggs in the fertilizable stage be broken 
up into fragments and these be inseminated, each fragment 
produces an embryo. The developmental capacity of such 
egg-fragments is as great in the egg of Chaetopterus, in which 
pluripotency is lost early during cleavage, as in that of 
echinoderm eggs, in which pluripotency persists longer. 
Some eggs normally give rise to many embryos, as that of 
the Texas armadillo that always forms four, and those of 
some insects which give rise each to hundreds. The fer- 
tilizable egg thus is a system of multiplex embryonic 
potency. This pluripotency however, except for normally 
polyembryonic eggs, is never realized in the normal process 
of development. Instead, the egg develops as a mono- 
embryonic system which becomes successively restricted 
within the domain of monoembryogenesis. It is in this 
domain that our problem of differentiation during cleavage 
lies. 2 

The primary event of the cleavage period is the act of 
cleavage, a rhythmical phenomenon. This sundering 
involves both nucleus and cytoplasm; for although in some 
cases the nucleus divides without cytoplasmic division at 
one time or another during cleavage, normally no egg ever 

1 For literature on merogony see Delage, 1S9S, 1899a and b. 

2 See also Just, I9^jb. 



develops throughout this period without cytoplasmic 
division. The furrowing of the cytoplasm, that is the act 
of cleavage itself, has been thought of as the cause of 
differentiation. This assumption however, is untenable, 
as I shall now show. 

By means of cleavage the cytoplasmic mass, the tgg y 
is successively sundered into smaller parts, the blastomeres. 
This sundering, even if regarded as a means for the separa- 
tion of constituents, does not bring about a new distribution 
of them by which arise embryonic parts. True, the divi- 
sion into smaller parts will facilitate distribution, but it 
can not be its cause. It is not the mere division into cells 
that makes the head, neck, arms of a human being or that 
distinguishes the liver as an appendage of the gut from 
which it is derived. Rather, concomitantly with the 
act of cleavage changes take place by which the embryonic 
areas subsequently arise. 

From yet another consideration we conclude that cleav- 
age as such is not differentiation. Even if we should 
assume that the uncleaved egg were an undifferentiated 
system, cleavage-partitions would only set off undifferen- 
tiated areas and the differentiation which arises during 
cleavage would be due to some other cause — as for instance, 
chemical reactions induced in a previously homogeneous 
mixture by the act of cleavage in setting up separate reac- 
tion-chambers of minute capillary dimensions. But these 
reactions and not the act of cleavage would then be the 
cause for differentiation. Or were we to suggest that by 
the establishment of cell-boundaries brought about by 
cleavage, the increase of surface renders possible surface- 
reactions characteristic of capillary spaces, we should still 
not support the proposition that the mere act of cleavage in 
sundering the protoplasmic mass is responsible for the pro- 
gressive differentiation which can be followed throughout 
the cleavage-process. 



Finally, consider the experimental evidence. This, 
although not embracing all eggs, indicates clearly that 
fertilized eggs may reach a certain degree of differentiation 
without cytoplasmic cleavage. 

Differentiation without cleavage has been noted by sev- 
eral observers to take place in eggs of annulated worms and 
of ascidians having been subjected to experimental treat- 
ment. The most thorough-going description of this type 
of development is that given for eggs of Chaetopterus having 
been exposed to hypertonic sea-water. 1 These eggs develop 
into most abnormal swimming forms made up of either 
uninucleated or multinucleated undivided cytoplasm due 
either to complete failure of cytoplasmic cleavage or to the 
disappearance of cleavages and fusion of the blastomeres. 

As development progresses, the cytoplasmic inclusions 
take up new positions in almost the same way and at the 
same time as in fertilized normally developing (untreated) 
eggs. But the ectoplasm behaves in a quite different 
manner. A portion of it moves to the animal pole, a 
smaller amount rests at the opposite pole. Later the ecto- 
plasm at the animal pole moves toward the vegetal pole 
so that the endoplasm is covered, thus simulating the over- 
growth of the larger yolk-containing blastomeres by the 
smaller ones in normal development. 

Another characteristic feature in differentiation without 
cleavage in the egg of Chaetopterus is the behavior of the 
nuclei in uninucleated eggs. In these eggs when the nucleus 
reaches the size of the germinal vesicle of the normal egg 
there appears a strong mutual attraction between it 
and the ectoplasm: the ectoplasm is drawn into the egg 
to form a mass that lies close to the nucleus or the chromatic 
part of the nucleus is drawn out to the ectoplasm. Eggs 
which show ectoplasm drawn into the interior do not 

1 Lillie, F. R. y 1906. 



develop as far as those in which the chromatic substance 
of the nucleus is drawn into the ectoplasm. 

We conclude therefore that although cleavage and dif- 
ferentiation normally go hand in hand, the relation is not 
one of cause and effect. 

The second event enumerated above as occurring during 
cleavage which has been held as a cause of differentiation, is 
the appearance of the embryonic axis and of the plane of 
bilateral symmetry, the so-called median plane. 

For egg-cells the term, polarity, means that with reference 
to an axis the egg in some way, as arrangement of pigment, 
location of the nucleus, site of polar body formation, etc., 
shows structural polar arrangement. Now it is often held 
that the polar axis of the egg bears some constant relation 
to the axis or the median plane of the embryo and on this 
assumption are elaborated theories of differentiation as 
determined by the egg's polar axis, of a differential axial 
gradient, and the like. 

In order to clear this problem, we must first distinguish 
between the polar axis in eggs of radially symmetrical ani- 
mals and that in eggs of the bilaterally symmetrical with 
respect to the axis of the embryo in the former eggs and to 
the median plane in the latter. Although this distinction 
is easy if only we appreciate certain simple geometrical 
truths, nevertheless we must give it prominence here 
inasmuch as so many writers have most unfortunately 
used axis and plane interchangeably in their theories of 

Polar and embryonic axes coincide in eggs whence radially 
symmetrical embryos arise. A line drawn through the egg 
from the site of polar body extrusion to the pole opposite 
is also the gastrular and the embryonic axis. In the eggs 
of these organisms — Porifera and Coelenterata — the gas- 
trula forms in such wise that its axis is along traces of the 
polar axis, no matter by which of the several modes of 
gastrulation that obtain in these eggs the gastrula arises. 



The gastrula may develop from a morula (a solid mass of 
cells) or a blastula (a mass of cells that contains a cavity); 
the gastrulation may be that of invagination, delamination 
or epiboly; finally the gastrula is a two-layered radially 
symmetrical structure enclosing a cavity whose polar axis, 
now the embryonic, for the most part traverses empty space, 
and at whose poles only are cells located which are part of 
the two-layered covering. Components of an axial gra- 
dient, if there be such, can become effective only in the 
cell-layer. 1 

Eggs of most bilaterally symmetrical animals begin their 
development as radially symmetrical structures and there- 
fore show a polar axis. But at the moment after fertiliza- 
tion when bilaterality appears in such an egg, we can no 
longer speak of an axis. In a bilaterally symmetrical 
organism — egg or adult — there exists no line common to 
planes as in a radially symmetrical one. Here, accurately 
speaking, we can use only the term, plane of symmetry. 

Certain eggs out of which develop bilaterally symmetrical 
animals — as, for example, those of Loligo, Hydrophilus, 
Amia — reveal bilateral organization before fertilization. 
In these, obviously, we can not speak of a polar axis. For 
even if we know that at some time in their history the eggs 
were radially symmetrical, the moment that bilaterality 
appears in them there can be no axis with respect to which 
the parts are symmetrically arranged. 

But even if we allow the incorrect use of the term, axis, 
in bilateral embryos, we find no constant relation between 
this axis and that of the egg. A survey of embryogenesis 

1 /. W . Wilson and I were never able to repeat Child' s observations 
on the effect of KCN on eggs of Arbacia which he interpreted as 
demonstrating an axial gradient. My own experiments (f$2Sc) 
on this egg, which gave results similar to Child's, strongly indicate 
that these are due to changes in the ectoplasm. Child's own work, 
as that on protozoan forms, can be explained in terms of effects on 
the superficial cytoplasm, 



in eggs whence develop bilaterally symmetrical embryos 
does not permit the conclusion that "the axis of the egg 
shows a definite relation to that of the gastrula, of the later 
embryo, and of the adult body"; and that "this relation, 
broadly considered, appears to be constant throughout the 
Bilateralia." 1 In many radially symmetrical eggs that 
subsequently show bilaterality it is true that a line drawn 
from the site where the polar bodies lie to the opposite 
pole is perpendicular to one drawn along the length of the 
embryo. In others, — e.g., the egg of Amphioxus 2 — the 
intersection of these lines forms an acute angle. In eggs 
that are bilaterally symmetrical before fertilization, the 
medium plane of the embryo lies in the egg's plane of bilat- 
eral symmetry. 

A survey of embryogenesis in the entire animal kingdom 
permits the conclusion that the embryo, with the possible 
exception of the mammalian, arises from the egg-surface. 
In the present state of our knowledge, the mammalian egg 
does not seem to fall in with our generalization, since its 
surface-cells form trophoblast. In all other eggs embryonic 
axis or median plane is normal not to the core of the egg 
but to its surface where the embryo lies. Axis or plane 
appears, due to new configurations in the protoplasmic 
system; 3 the embryo-axis or the plane of bilaterality is an 
expression and not the cause of the differentiation which 
unfolds itself as cleavage progresses. 

We turn now to another of the events occurring during 
cleavage which were enumerated above, the shift of 
the cytoplasmic inclusions, again seeking the cause of 

1 Wilson, 1924. 

2 Cerfontaine. 

3 One such configuration is undoubtedly set up by the entrance of 
the spermatozoon into the egg — see Just, IQI2. 



The positions especially of the oil and the yolk in the 
egg-plasma change after fertilization, as comparisons of the 
unfertilized with the fertilized eggs of either Nereis or 
Chaetopterus reveal. In the egg of Arbacia as of any other 
echinid the shift in oil and yolk is not so well marked. 
That is, in eggs with determinate cleavage there is a more 
clear-cut change of the cytoplasmic inclusions to new posi- 
tions than in indeterminately cleaving eggs. The progres- 
sive differentiation from the single cell, the egg, to a complex 
organism, the embryo, was once thought of as due to the 
distribution of the visible materials in the cytoplasm: 
embryo-formation was ascribed to the segregation of these 
fat- and fat-containing materials which were therefore 
denominated organ-forming substances. To-day, we can 
appreciate the naivete of this theory, wondering how we 
could have given it such serious consideration. 

Eggs differ 1 with respect to content of cytoplasmic inclu- 
sions. This is especially true of yolk. Indeed, eggs are 
sometimes classified on the basis of yolk-content. Once 
we spoke of yolk-less (alecithal) eggs; now we know that 
all eggs, even the human, contain yolk. Thus it is better to 
classify eggs not as yolk-free and yolk-rich, but rather as 
telolecithal (yolk in one part), centrolecithal (yolk in the 
center) and homolecithal (yolk evenly distributed). Yolk 
also differs physico-chemically in different eggs; and I have 
some evidence which indicates that it differs in a given egg 
before and after fertilization. What is true of yolk, the 
food material for the embryonic organism, is true of mito- 
chondria, pigment-granules, etc.: they vary as to amount, 
distribution and physico-chemical make-up. Whilst all 
of these cytoplasmic inclusions of which I here speak differ 

1 The reader will have noted that I often emphasize the differences 
in animal eggs. In this my general purpose is to sift from the 
differences that which is similar and equivalent and thus to order 
and to define the biological problems. 



from each other, they are, so far as I know, wholly or in part 
composed of lipin (fat or fat-like substance). Though there 
is no reason against the postulate that the organ-forming 
substances are composed of fats, nevertheless, I believe 
that we are on unsafe ground in attempting to relate the 
formation of the many various organs to fat-containing 

Sometimes the cytoplasmic inclusions show differential 
distribution to very definite blastomeres. Consider the 
egg of a low form of chordate, for example, that of the tuni- 
cate, Cynthia. One substance (yellow) maintains a defi- 
nite distribution to given blastomeres. This is an egg 
with determinate cleavage. From the blastomeres contain- 
ing the yellow substance always in the normal egg muscle 
and mesenchyme develop. Apparently, therefore, here is 
a very clear case of an organ-forming substance definitely 
localized in the egg before first cleavage. But experimental 
findings do not permit such a conclusion. To these I shall 
later turn. 

Other eggs with determinate cleavage for the most part 
do not exhibit prelocalization so clearly as that of Cynthia, 
though their cytoplasmic inclusions shift from their original 
positions before fertilization to new ones after fertilization 
and at first cleavage; thus the yolk and oil come to lie in 
cells which are destined to form the gut. This is not evi- 
dence that oil and yolk "determine" gut-formation. Also, 
there are many other eggs that do not show any special or 
constant disposition of the cytoplasmic inclusions. 

On other grounds the theory that the visible cytoplas- 
mic inclusions are organ-forming substances is untenable. 

In the first place, I call attention to the egg of Strongylo- 
centrotus lividus, a sea-urchin commonly found in the 
Mediterranean, which may exhibit a beautiful superficial 
band of orange pigment below the equator in the hemisphere 
opposite the animal pole. Boveri used the pigment band 



of this egg as a means of orientation in experiments. But 
the occurrence of this band is inconstant; not every speci- 
men of Strongylocentrotus gives eggs containing it. There 
is no virtue in colored particles as such suspended in the 
cytoplasm for differentiation, though they may be useful 
indicators in other respects. 

Another cogent argument against the theory that the 
visible cytoplasmic inclusions are organ-forming substances 
lies in the fact that cleavage in many eggs may ensue for 
several cell generations apart from the underlying yolk and 
oil; this is true of all eggs with discoidal cleavage. In them 
the cytoplasm is at first a transparent and apparently 
homogeneous sheath and later a disc below the vitelline 
membrane. Cleavage is confined to this inclusion-free 
cytoplasm. When in later stages of development — after 
the differentiation occurring during cleavage has taken 
place — the embryo grows at the expense of the yolk-laden 
region of the egg, the yolk is transformed into clear cyto- 
plasm. In other words, in these as in all animal eggs, 
yolk and oil play no direct part in differentiation. 1 

Let us recall another fact: many living eggs under the 
microscope appear to be transparent. Rigorously to adhere 
to the theory of visible organ-forming substances is to deny 
organ-formation in transparent eggs. By ascribing a 
primary role to the visible inclusions in the cytoplasm, we 
have had the tendency to reckon the flood by its burden, 
taking the traffic for the stream. 

Finally, certain experimental results set up an unsur- 
mountable obstacle to acceptance of the theory of organ- 
forming substances as an explanation for differentiation. 

Above I have pointed out that due to the ectoplasmic 
changes caused by fertilization, eggs exhibit cytoplasmic 

1 In trematode eggs exists the strongest separation between egg and 
yolk, because the egg alone is a product of the ovary; the yolk is 
produced by accessory glands and spun around the egg. 



currents which move the inclusions. This movement is 
most striking in eggs with determinate cleavage — especially 
if the inclusions be colored and hence easy to identify. 
They come to lie constantly in definite regions of the egg 
at first cleavage. If these fixed positions of the inclusions 
be responsible for differentiation, then altering them should 
modify or hinder development. 

By centrifugal force the normal positions of the cytoplas- 
mic inclusions can be altered. The materials, which as 
spherules and granules are suspended in the cytoplasm 
normally without reference to their specific gravity, are 
when the eggs are centrifuged moved to new positions and 
stratified according to their respective specific gravity. 
Thus oil, yolk and pigment form layers in the order named 
with a zone of clear cytoplasm beneath the oil. Here our 
interest focuses on two results obtained from the centri- 
fuging of eggs. 1 

The first deals with the fact that centrifugal force in 
altering the position of the inclusions does not alter the 
direction of the cleavage planes. Hence, the new arrange- 
ment of the inclusions induced by centrifugal force, which 
shifts them to blastomeres in which in normal development 
they would not come to lie, has no effect on the develop- 
ment of the embryo. It should be emphasized that this 
effect of centrifugal force is the same in eggs with deter- 
minate and indeterminate cleavage. Investigators are 
agreed that the altering of the position of the cytoplasmic 
inclusions has no effect on embryo-formation : the eggs 
develop normally although the inclusions come to lie in 

1 Although centrifuging may be a violent method of treating eggs, 
capable of causing their destruction^ it can^ if used with discretion, 
leave them quite uninjured. One can soon learn the least amount 
of centrifugal force necessary to stratify the cytoplasmic materials; 
there is here no point in using a greater, as Conklin did on the 
ascidian egg. 



blastomeres other than those that normally hold them. 
For the cleavage planes bear any relation to the axis of 

Even more significant is the second result, namely, that 
fragments of clear inclusion-free cytoplasm taken from 
centrifuged eggs, whether these eggs cleave determinately 
or indeterminately, develop when fertilized as whole eggs. 
Many unfertilized eggs — e.g., those of sea-urchins and those 
of other species whose germinal vesicles have broken down 
■ — can be strongly deformed by pressure, centrifugal force, 
etc. This is due to the fact that the fluid protoplasm is 
enclosed by a highly elastic membrane. Such eggs when 
centrifuged are pulled out into strands, a fact known already 
many years ago. With a greater degree of centrifuging 
the strands break up into fragments. Some of these frag- 
ments, with or without the egg nucleus, are composed of 
the clear hyaline cytoplasm only. Such clear fragments 
devoid of inclusions are capable of fertilization and develop- 
ment; they cleave as whole eggs. 1 

These two experimental findings alone render untenable 
the theory that considers the visible inclusions as organ- 
forming substances. The differentiation from the single 
cell, the egg, to the complex multi-cellular embryo can not 
be related to the distribution of visible materials suspended 
in the cytoplasm. That these materials have functions, 
no one would deny. Most of them, oil and yolk, are food 
for the embryo or larva; the remainder are doubtless for 
respiration, secretion, etc. They move with the cyto- 
plasmic ebb and flow but are not themselves the tide of life. 

The orderly progressive shifting of the visible multi- 
colored spherules and granules in a living egg, especially 
in one with determinate cleavage, is an entrancing spec- 
tacle: in defiance to gravity, oil, yolk, mitchondria and 

1 Lillie, F. R., igo6. 



pigment granules, each at a different rate, shift to new posi- 
tions, always the same in every undisturbed egg of a given 
species and definite at its every cleavage. In the order 
and amplitude of this shifting we recognize the power of 
the so little known cytoplasmic currents. Since these in 
turn are directed by ectoplasmic changes, as I have shown, 
we may by study of the shifting of the inclusions come to 
know more about the ectoplasm and its interaction with 
the inner cytoplasm. 

In this inner cytoplasm is situated that fixed and little 
changing cell-component, the nucleus, to which has been 
assigned the role of maintaining intact the inheritance of 
the species. What is its role in differentiation? 

Along with the successive acts of cleavage by which the 
egg substance is divided up into blastomeres and as the 
embryo-axis or plane of symmetry becomes recognizable, 
the original single nucleus of either the fertilized or the 
parthenogenetic egg undergoes division so that each blasto- 
mere receives a nucleus. In discussing the probable role 
of the nucleus as a causal factor in the differentiation during 
cleavage, I shall now deal with the following points: the 
quantity of nuclear material finally found at the close of 
the cleavage-period, the origin of this material, and how 
the nuclei, with special reference both to chromosome- 
content and to the genes making up the chromosomes, may 
be conceived as taking part in determining the development 
of the egg. 

Although with each successive nuclear division during 
cleavage of the egg the nuclei progressively diminish in 
size, no individual nucleus in any blastomere at the end of 
cleavage ever vanishes. On the other hand, the total 
quantity of nuclear Substance, where this has been deter- 
mined, is greater at the end than at the beginning of the 
cleavage-period. The quantitative increase in nuclear 
substance continues through the whole embryonic period 



and becomes therefore a definite characteristic not onlv 
for cleavage but also for the whole course of develop- 
ment. The body of a newly hatched chick contains 
nuclear matter far in excess of that in the uncleaved 
egg which gave rise to the animal. Indeed, as long as the 
chick lives and is capable of producing germ-cells it elab- 
orates nuclear substance. Moreover, every organism that 
regenerates lost parts, every one whose tissues exhibit 
pathological growths, as tumors, builds nuclear substance. 
Hence, the building up of the chemical constituents of the 
nucleus represents a basic property of protoplasm as a self- 
reproducing system. In other words, as protein-synthesis 
represents that fundamental chemical characteristic of 
living matter that distinguishes it from non-living, so 
nuclear synthesis (in part a protein-synthesis also) stands 
as one of the primary chemical activities of the self-regula- 
tion and the self-diff erenti ation exhibited as special attributes 
of cells capable of reproducing themselves. The course 
of development is marked by syntheses. By these arise 
secretions as those of the thyroid, the pancreas, etc., and 
such compounds as haemoglobin, the iron-containing respir- 
atory pigment of the blood, as found in the embryos of 
animals which possess these substances. By synthesis also 
nuclei arise. 

The question now is: out of what are the nuclei synthe- 
sized? With the progressive increase in amount of nuclei 
runs during the period of cleavage a decrease in that of the 
cytoplasm without any change in the amount of the yolk 
and oil. This fact, holding for all animal eggs, is especially 
clearly shown in totally cleaving eggs. In the egg of Nereis, 
for example, I have followed very exactly the composition 
of the blastomeres throughout cleavage and can state that 
in the blastula-stage the total amount of cytoplasm is 
less than that in the egg at first cleavage, whereas the oil 
and yolk do not change in amount. The clearest evidence 



that nuclei arise out of the cytoplasm is furnished by the 
development of those clear hyaline pieces of protoplasm 
already referred to, for in the cleavage-process of such frag- 
ments also the nuclear matter increases and the cytoplasm 
decreases. It has been shown that the nuclei in yolk- 
free blastomeres are larger than those in yolk-rich blasto- 
meres and that the size of the nuclei is proportional to the 
hyaline content of a blastomere rather than to the total, 
encompassing the inclusions. 1 These facts prove that in 
totally cleaving eggs during cleavage the nuclei are synthe- 
sized from the cytoplasm. In partially cleaving eggs as well 
the yolk takes no part in development during cleavage. 
When, as happens in these eggs, at the end of cleavage new 
cells are added to the embryonic area from the yolk region, 
the yolk, as the evidence indicates, is transformed into cyto- 
plasm and does not go directly into the nucleus. 

The increased nuclear content at the end of cleavage 
therefore can only mean that the nuclei are elaborated out 
of nuclear stuffs or their precursors; it can not mean an 
automatic activity of the nucleus capable of building nuclei 
out of nothing. Nuclei are built up from the cytoplasm. 
So too their constituents, the chromosomes. 

In preparation for fertilization both egg and sperm-cell 
go through a process by which the number of chromosomes 
characteristic for the species is halved; at fertilization this 
number is restored, half from the father, half from the 
mother. This is the strongest evidence in favor of the 
chromosomes as the means by which the off-spring inherits 
qualities from both parents. The number of chromosomes 
of the fertilized egg is the somatic or so-called diploid 
number characteristic for the species. During development 
this number remains constant. Every somatic cell of the 
adult organism no matter how complex contains in its 

1 Conklin, IQI2. 


nucleus the number of chromosomes equal to that of the 
fertilized egg. The germ cells of the adult in their terminal 
stages after the process of reduction again possess half the 
somatic number. Thus the cycle from germ cell to germ 
cell is complete. 

During the cleavage stages, the chromosomes are halved 
in quantity at each cell-division. Since, as we know, they 
never reach the zero-point, they must increase in mass 
during the period of cleavage. Says Morgan 1 : ,c During 
development, especially during early cleavages, the amount 
of chromatin steadily increases in amount, giving an expo- 
nential curve resembling the first half of a curve of a mono- 
catalytic reaction/ 5 What evidence we possess definitely 
warrants the statement that the chromatin material 
increases in amount during cleavage. 

Suppose we consider the egg of a large fish, a carp or 
salmon. Such an egg after fertilization has two groups of 
chromosomes, one from the sperm-nucleus and one from 
the egg-nucleus, which united give the total number of 
chromosomes characteristic for this particular species of 
carp or salmon. Eventually from this single fertilized egg 
the adult fish develops, which is composed of millions and 
millions of cells constituting all of the organs of the fish; 
if it is a female, it produces thousands or even millions of 
eggs, if it be a male, billions of spermatozoa. Eggs or 
spermatozoa of an adult fish have chromosomes equal in 
number, size, form and, presumably, in weight to those 
found in the egg- or sperm-nucleus of the fertilized egg from 
which this adult fish was developed. 

If the chromosomes grow — and it is inconceivable that 
they do not — they grow at the expense of something; they 
can not grow directly out of themselves without obtaining 
the new materials for themselves. The synthesis of chro- 

1 Morgan, 192J. 



matin demands the utilization of raw materials out of which 
chromatin is constituted. Then the question arises: Is 
chromatin synthesized directly from the elements of 
which it is composed or from compounds? We know 
that the synthesis, upon which directly or indirectly all 
living stuff depends, comes from CO? and HOH which in 
the presence of chlorophyl and sunlight make the simplest 
sugars. From these by polymerization polysaccharides are 
derived and, with N, proteins and fats are built up. In 
other words, the protoplasmic synthesis of organic com- 
pounds like proteins is never from elements directly but 
from compounds in the cell. If such protoplasmic syn- 
theses are from compounds, why not chromatin, itself a 
conjugated protein? Chromatin grows because more of 
it is made; and it is made from compounds available in the 
cell. Even if we assume that it is made out of that material 
which is already in the nucleus as non-chromatin, we must 
conclude that this non-chromatin in being part of the 
nucleus has come from the cytoplasm. 

It is clear, first, that chromatin grows; second, that it 
can not grow in some magical way but most probably 
grows as other cell-stuff grows — i.e., according to principles 
governing protoplasmic syntheses as far as we to-day know 
these, that is, from preexisting and simpler compounds. 
The fact that during the cleavage stages of most eggs the 
cytoplasm decreases as the chromatin increases further 
indicates that chromatin is synthesized directly out of the 
cytoplasm. Oil and yolk as such play no part in this 
process since they tend to be segregated and the cells con- 
taining them are the least actively dividing and those free 
of them most productive in the elaboration of new chro- 
matin substance. 

Thus we deal with the definite increase of a chemical 
stuff, nuclein; a measured weight of this must mean a 
definite weight of precursors that produce it. Briefly, in 



dealing here with this conversion of cytoplasm into nuclei 
we are concerned with the formation of a known chemical 
stuff. If now we relate differentiation to this chemical 
process, a process whereby a definite chemical stuff is 
removed from the cytoplasm so that cytoplasmic reactions 
are rendered possible, we may have close at hand an 
explanation of differentiation as a series of chemical changes. 
In this wise it may be possible to detect certain chemical 
stuffs as potencies. Surely, the more we can substitute for 
such terms as "potency'" chemical reactants in chemical 
reactions, the closer shall we come to the solution of the 
problem of differentiation of development. In relating 
differentiation in part to the synthesis of nuclear material, 
we take the first step in this direction. 

The progressive differentiation of the egg during cleav- 
age according to this conception is brought about neither 
by the pouring out of stuffs by the chromosomes into the 
cytoplasm nor by segregation of embryonic materials as 
postulated by those who uphold the theory of embryonic 
segregation, but by a genetic restriction of potencies 
through the removal of stuff from the cytoplasm to the 

Consider a fertilized egg, ABCD, with determinate cleav- 
age, which at first cleavage forms two blastomeres, AB and 
CD; there must be differentiation, since the AB and the 
CD blastomeres when separated give rise to partial larvae. 
This would mean according to the theory of segregation 
that AB is minus CD's material and CD is minus AB's. 
AB thus would be a cell in which the prospective ^^-poten- 
cies are present; the same would be true in the CD cell for 
the CD-potencies. After the second cleavage, A-^ i?-, C- 
and D-potencies would be present in blastomeres A, B, C 
and D respectively. Similar segregation would happen at 
each succeeding cleavage. For eggs with indeterminate 
cleavage the presence of material — and conversely, the loss 

3 T 3 


of other material — would take place at that stage in the 
cleavage-process when the blastomeres on separation lose 
the potency for development into complete though dwarf 
larvae. But is an egg-cell (in this case with determinate 
cleavage) ABCD = AB + CD, as we would express it 
according to the theory of segregation ? Or can we say that 
AB = AB + (-CD); CD = CD + {-AB)} Can we say 
that ABCD is really AB + (-CD) + CD + (-AB) and do 
we gain thereby ? 

Restriction is, as I shall show, preferable to segregation 
for it more nearly expresses the process. Restriction 
implies a loss, in this case of developmental potencies, 
without necessarily a rearrangement of materials. Segre- 
gation connotes new arrangement or sorting out of mate- 
rials without any implication of change in them. If as 
cleavage progresses cytoplasmic materials for embryo- 
formation are merely shifted to new positions, then reversal 
of the cleavage process should be possible. Stages of dif- 
ferentiation then would be repetitive. I do not wish to 
indulge in hair-splitting definitions; nevertheless I must say 
that I for my part can not see how the materials of an egg by 
mere acts of separation can account for differentiation. 
Against the assumption of a mere transposed order of 
materials stands the fact that as development proceeds the 
individual blastomeres lose potency. 

At first cleavage blastomere AB becomes such because 
it loses CD-material, and blastomere CD becomes such 
because it loses ^/i?-material. A separation into AB- 
and CD-blastomeres means that AB somehow removes 
all CD-material and CD all .//immaterial. So does gene- 
tic restriction begin and so it continues. This takes 
place through the absorption of CD-material by the 
nucleus of the ^/i?-blastomere-to-be and of the A ^-material 
by the nucleus of the CD-blastomere-to-be. The subtrac- 

3 J 4 


tion of the cytoplasmic materials by the nucleus takes place 
with each mitotic cycle. 

We know, as has been pointed out, that as cleavage pro- 
gresses the nuclei increase in number and en masse in vol- 
ume. That is, they grow. We know also that in this same 
period the total volume of cytoplasm decreases and that the 
growth of the nuclei is at the expense of the cytoplasm. 
Genetic restriction then depends upon the removal by the 
nucleus of certain materials from the cytoplasm, leaving 
others free. The free materials determine the character of 
the cell — as ectoderm, mesoderm or endoderm and later as 
any one of the organs arising therefrom. 

Something leaves the cytoplasm with each cell-division; 
then why not the materials of the cytoplasm which are non- 
specific for the given blastomere? Certainly, the mecha- 
nism for the removal of material is present in this growth 
of the amount of nuclear substance as development pro- 
gresses. With each cleavage each nucleus fixes all material 
other than that which makes the blastomere what it is, 
AB or CD] A or B, C or D, etc., to the end of cleavage. 
Then the nucleus of cell AB is different from that of the 
CD cell since the y/5-nucleus contains bound CD-material 
and vice versa for the CD-nucleus. 

Then the potencies for embryo-formation are all present 
in the uncleaved egg; cleavage serves to remove these and 
this removal is fast or slow, early or late depending upon 
the species of egg. The question arises: When does the 
cytoplasm of the egg gain its potencies for differentiation 
during cleavage ? 

Pluripotency becomes demonstrable through merogony, 
as was pointed out, with the onset of the fertilizable condi- 
tion inasmuch as now fragments of the egg are when fer- 
tilized each capable of development. From this we might 
reason that the potencies that were stored up in the nuclei 



during the previous development of an egg are restored to 
the cytoplasm at this time, that is, to the cytoplasm of 
the new egg. However, the demonstration that pluri- 
potency is present gives no evidence of the time when it 
arose. Merogony may be only an indicator of an earlier 
established pluripotency. Having no other criterion than 
fertilizability for the demonstration of pluripotency, we 
can not exactly define the moment when the egg becomes 

An attractive hypothesis is that pluripotency arises with 
breakdown of the germinal vesicle when there escapes into 
the egg-cytoplasm residual nuclear stuff greatly in excess 
of what remains to form the mitotic complex of the first 
maturation-division. The potencies might be regarded as 
identical to, or associated with, this extra-nuclear material. 
According to this suggestion, the rise of pluripotency would 
be separated from the fertilizable period, for the fertiliz- 
ability does not depend upon a particular stage in matura- 
tion. But since many species of eggs are fertilizable whilst 
their germinal vesicles are intact, not a single one of these 
should be pluripotent if we are to relate the rise of pluri- 
potency to substances escaping into the cytoplasm after 
breakdown of the germinal vesicle. Where, however, frag- 
ments taken 1 from such eggs develop when fertilized, it 
must be ascertained that the fragments are devoid of stuff 
having escaped from the germinal vesicle. The lack of 
data on merogony in such eggs does not warrant a decisive 
conclusion in these questions. 

There remains another possibility. After the germ-cells 
have become differentiated from somatic cells through the 
loss to their nuclei of all potencies, with only the potencies 
for germ-cells left free in their cytoplasm, they become 
isolated from the soma. This isolation brings about the 
escape of all potencies that were up to that time bound in 
the nuclei, into the cytoplasm. Thus the eggs would 



become pluripotent. The condition of the spermatozoon 
is discussed later. 

Any theory offered to account for the differentiation that 
takes place during cleavage should be consistent for both 
the cleavage-period and the succeeding periods of develop- 
ment during which differentiation occurs, since in essen- 
tials, every period of differentiation is alike. 

Certain phenomena occurring in the organism as egg, as 
cleaving mass, as embryo and as adult are based on some 
differentiation. A theory of differentiation during cleav- 
age should hold for these phenomena. They are: (i) 
polyembryony and experimental twinning; (2) merogony; 
(3) the development of diploid fragments; (4) the develop- 
ment of isolated blastomeres; (5) haploid parthenogenesis; 
(6) experimental and natural polyploidy; (7) asexual repro- 
duction by budding and fragmentation, and alteration of 
generations; (8) regeneration of lost parts; and (9) the origin 
of tumors. I shall now endeavor to show that these phe- 
nomena are better explained on a theory of differentia- 
tion as genetic restriction than on one that postulates 
segregation. 1 

1. The capacity of some eggs, as those of certain insects, 
normally to produce many embryos from one egg as well as 
the possibility of producing twins experimentally from 
normally mono-embryonic eggs including those of the 
determinate type, as the eggs of Nereis, Chaetopterus, 
Tubifex y etc., shows that eggs have more latent potency 
than that required for producing one animal. A theory 
postulating a restriction of potencies seems to meet these 
facts better than one suggesting that materials for the 
embryo are segregated. 

1 Cf. Perez (19 12) who lists much the same experimental evidence 
against the mosaic-theory (an earlier form of the segregation-theory) 
of development, which he justly considers a modern version of the 
preformation doctrine. 



2. This same conclusion holds for the data on merogony. 
It may be regarded as proved that all eggs which can be 
fragmented before fertilization have the capacity for the 
production of many embryos. Merogonic development 
indicates strongly, especially in determinate eggs, that 
differentiation ensues as a restrictive process — restricting 
potency for multiple embryo-formation to one. The theory 
of segregation in these cases would imply telescoping of 
several embryos. 

3. Some fragments of eggs contain the egg-nucleus and 
hence when fertilized develop with two nuclei constituting 
a diploid nucleus. In such fragments restriction obtains 
as in whole eggs, except that the chromosomes remove less 
cytoplasmic stuff since less is available. From the point 
of view of embryonic segregation, we would have to assume 
that since perfect though dwarfed embryos result from the 
fragments, the segregates in whole eggs are pluralistic. 
From this it should follow that all eggs when separated into 
blastomeres should develop into entire organisms or into 
embryonic regions, each containing parts reduplicated. 

4. The development of blastomeres isolated during 
cleavage into perfect though dwarf embryos stands as 
strong evidence that restriction and not segregation under- 
lies differentiation. For how from already segregated 
areas could perfect embryos arise ? Or, if it be postulated 
that the areas are not yet segregated at the stage of isola- 
tion, why do the blastomeres in an intact egg not develop 
each into an embryo? On the basis of our theory of 
restriction, potencies bound in the cytoplasm in the intact 
egg become free in the isolated blastomeres because of the 
new conditions set up by isolation. 

5. If we put forward the theory of segregation to account 
for the differentiation occurring in a parthenogenetic egg 
containing a nucleus with half the number of chromosomes 
found in the eggs of this species when they are fertilized, 



we encounter the difficulty of explaining how under the 
domination of this "half" nucleus the cytoplasm is ordered 
into regions whence the organs arise. The theory of restric- 
tion by means of the nucleus removing potencies from the 
cytoplasm does not encounter this difficulty for accord- 
ing to it the nuclei extruded as polar bodies relieve the 
cytoplasm of potencies. 

6. Similar reasoning applies to eggs developing with 
polyploid nuclei — i.e., with more chromosomes than those 
contained in the united egg- and sperm-nuclei. In the egg 
of Nereis, for example, fertilized after treatment with 
ultra-violet light, 1 the polyploid nucleus, made up of the 
three nuclei from the suppressed polar bodies plus the egg- 
and the sperm-nuclei, remove cytoplasmic potencies pre- 
cisely as these are removed in normal fertilization by polar 
bodies and egg- and sperm-nuclei. But from the point of 
view of segregation, conditioned by nuclear material or 
power escaping into the cytoplasm, development should 
not take place because of the over-powering influence of 
the super-abundant nuclear matter. 

7. Certain eggs develop into animals which have the 
capacity for asexual reproduction by the formation of buds 
or by the process of breaking up into two or more fragments. 
Both the buds and the fragments develop into complete 
organisms similar to the form whence they arose. If sexual 
and asexual phases regularly succeed each other, they con- 
stitute a life-history said to reveal alternation of generations. 

Asexual reproduction can be explained by assuming that 
potencies in the egg, present in excess of those for the forma- 
tion of a single individual, remain free. The reduplication 
of the animal by bud or fragmentation though it occurs 
late in the life-history is thus comparable to poly-embryony. 
In those cases of alternation of generations, where egg- and 

1 Just, 1933c. 



sperm-producing areas as motile swimming organisms 
arise, restriction has taken place by removal of these free 

8. Regeneration of lost parts is encountered in all ani- 
mals; in some, as the flatworms, it reaches the level of 
asexual reproduction; in others, capacity for regeneration 
is limited. In some forms regeneration is said to be due 
to the presence of formative cells, cells in which it is assumed 
that differentiation has been arrested; in others regenera- 
tion of structures occurs from tissues that normally never 
give rise to them. 1 In either case the capacity for regenera- 
tion may be thought of as a reorganization under the influ- 
ence of the liberation of potencies, previously bound by 
the chromosomes, into the cytoplasm induced by the 
changed condition of the cells as a result of the injury or 

9. Abnormal growths or tumors can be explained in the 
same way. Some change in the environment of the cells 
stimulates the throwing out by the nuclei of potencies into 
the cytoplasm where a new type of development is set up. 
A tumor-cell is one which has escaped the domination exer- 
cised by contiguous cells. It becomes physiologically 

In view of all of these facts that support my theory of 
differentiation as genetic restriction, I state this again: 
As development progresses, the egg-potencies are restricted 
through their withdrawal from the cytoplasm by the 
chromosomes with each successive cell-division. Thus the 
cytoplasm forms functional areas. At some time in the 
history of the egg's development the potencies, having 
been previously taken out and stored by the chromosomes 
during cleavage and succeeding stages of differentiation, 
escape into the cytoplasm. The cytoplasm of the fertilized 
or parthenogenetically developing egg restores them again 

1 Ree^ 1904. 



to the chromosomes. This theory of differentiation as a 
genetic restriction is consonant with the nine phenomena 
enumerated above. 

This conception of the role of the chromosomes in the 
process of differentiation stands in contrast to the attempt 
of genetics to explain differentiation. 

The last thirty years have seen as an outgrowth of the 
well-known Mendelian laws that flourishing branch of 
biology, genetics, nourished by the knowledge of chromo- 
some behavior, develop almost to the proportions of a 
separate science — at least it has a very rich vocabulary of 
its own. Mendel's laws are not causal, but statistical con- 
clusions concerning the regularity with which offspring 
show the characters of parents. The chromosome theory 
of Mendelian heredity attempts a causal explanation. 

According to the current conception each chromosome is 
a string of units, the genes, which are the carriers of hered- 
ity. The gene theory has been developed by work done on 
the small fruit-f[y y Drosophila y raised in the laboratory under 
fairly constant conditions of temperature, of food, of per- 
iodic subjugation to heavy doses of ether, etc. Thus 
divorced from the rigors of nature these animals imprisoned 
in large numbers in small glass containers have undergone 
changes which endure for so many generations that they 
are without doubt constant. Animals exhibiting these 
enduring changes are mutants. For each mutation genetics 
has assigned a place on one or another chromosome; some- 
times determiners of the same mutation are located on 
several chromosomes. Among geneticists the gene-theory 
is widely accepted. Among biologists, on the other hand, 
even of those who do not accept the gene theory of the 
geneticists, the majority agree that the chromosomes are 
the bearers of heredity. 

According to the geneticists the chromosomes in every 
cell of the most complex organism are identical with those 
of the fertilized egg. That is, as they express it, the chro- 



mosomes maintain their individuality. The linear arrange- 
ment of the genes in each chromosome 1 is the same for the 
given chromosome whether it is in a fertilized egg, in a kid- 
ney-cell, a muscle-cell or any other cell of the adult organ- 
ism. Now at each cell-division though the chromosomes 
are split they must somehow maintain their identity or 
individuality; this maintenance is the prevailing doctrine 
among geneticists. 

Speaking of the individuality or genetic continuity of 
the chromosomes, Wilson says 2 : 

In any general account of the history and genetic relations 
of the chromosomes in the life-cycle, we inevitably find our- 
selves speaking of them as if their identity were really lost 
when they disappear from view in the resting or vegetative 
nucleus. The vast literature of the subject is everywhere 
colored by the implication that chromosomes, or something 
which they bear, have a persistent individuality that is 
carried over unchanged from generation to generation. This 
view has met with some determined opposition; but with the 
advance of exact studies on the chromosomes escpticism 
has gradually yielded to the conviction that the chromo- 
somes must, to say the least, be treated as if they were per- 
sistent individuals that do not wholly lose their identity at 
any period in the life of the cell but grow, divide and hand 
on their specific type of organization to their descendants. 
This does not mean that chromosomes are to be thought of 
as fixed and unchangeable bodies. Beyond a doubt they 
undergo complex processes of growth, structural transfor- 
mation and reduction, in some cases so great that no more 
than a small fraction of the substance of the mother- 
chromosomes at its maximum development is passed on to 
the daughter-chromosomes. Whether we can rightly speak 
of a persistent "individuality" of the chromosomes is a 
question of terminology. What the facts do not permit us 
to doubt is that chromosomes conform to the principle of 
genetic continuity; that every chromosome which issues 
from a nucleus has some kind of direct genetic connection 

1 Delage^ /£<?5, p. 751 ', very clearly expressed the idea of a linear 
arrangement in the chromosomes. 

2 Wilson, 1925. 



with a corresponding chromosome that has previously 
entered that nucleus. 

What here is individuality? If I am able to keep alive 
all the descendants which arise from a single slipper ani- 
malcule by cell-divisions at the rate of three divisions a day, 
in seven days I shall have 2,097,152 organisms, each of 
which has 1/2,097,152 part of the original Paramoecium. 
Only this fraction, then, of individuality is maintained. 
And this is a much simpler case than that of individuality 
of each member in a chromosome-complex from fertilized 
egg to adult of a complex organism. I think that the 
question of the persistent individuality of the chromosomes 
is more than a question of terminology. 

If we turn from the chromosomes to the genes themselves 
that compose each chromosome, we find the same difficulty. 
It has been suggested that the gene is a molecule. The 
two daughter-cells arising from first cleavage would then 
possess chromosomes made up of half molecules; in the four- 
cell stage there would be quarter molecules; and in each 
succeeding division there would be a corresponding reduc- 
tion of the original gene-molecules found in each chromo- 
some of the fertilized egg. But in chemistry Ave know of no 
fractional molecules. Then, let us start with the adult 
organism. On the assumption that each gene in each 
chromosome of every cell of the whale, for example, is a 
molecule, it is necessary to assume that each gene of the 
fertilized egg is a complex of molecules whose number 
equals that of the sum total of all the descendants from 
this gene which are in each and every cell of the adult. It 
would be necessary further to assume that the gene-mole- 
cules vary in size in stages of development from egg to 
adult. The gene therefore can not be a molecule according 
to physico-chemical usage. The proponents of the gene 
theory should be the last to postulate any non-physico- 
chemical molecule. 



Thus the term, individuality of the chromosomes or of 
their constituent genes, is of restricted meaning. I can 
not see how a given chromosome or a gene in the fertilized 
egg should be the same individual in every cell of the body 
of an adult male including every one of the billions and 
billions of spermatozoa that such an animal, like the trout, 
for example, sheds during its life-time. With regard to the 
"individuality" of the genes or the chromosomes genetics 
has offered us nothing except a term, which, as we have 
seen, is not even clear. From this point of view the gene 
theory certainly can not give an explanation of the develop- 
ment of an egg into an adult organism. 

Let me hasten to state that in company with the majority 
of biologists I consider the weight of evidence sufficient 
for the assumption that the chromosomes have to do with 
heredity. As I have pointed out already, I see in the 
nucleus that component of the cell which tends to maintain 
the specificity of the cells by its change-resisting character. 
Then the chromosomes maintain individuality or identity. 
This maintenance however, as we have seen, can only come 
through growth at the expense of the cytoplasm. This fact 
allows us to see in a new light this maintenance : it is brought 
about by growth, the up-take of material that carries or 
assumes the characteristics of that which is already present 
in the chromosome. Further, this maintenance of identity 
must stand in some relation to the source that furnishes 
the material, namely, the cytoplasm. 

Counter to this fact of the growth of the chromosomes 
at the expense of the cytoplasm runs the postulate of 
those geneticists who seek to explain differentiation 
by the gene-theory of heredity, namely, that the genes 
are factors which order the developmental processes 
by giving up substance to the cytoplasm. But even 
if the upholders of the gene-theory of heredity accept 
the fact that the chromosomes grow at the expense of 



the cytoplasm, their conception of the action of the genes 
as unalterably fixed entities can not explain differentiation 
of development. For how could the genes be responsible 
for differentiation, if they are the same in every cell? 1 
Unless the geneticists assume that their genes are omnipo- 
tent, we can not understand how the problem of differentia- 
tion can be solved by the gene-theory of heredity. 2 

The gene-theory of heredity is an ultra-mechanical rig- 
orously bound concept. This mechanistic rigidity renders 
it inadmissible as an explanation of the process of develop- 
ment, a process marked by the egg's inherent capacity and 
by its mobile responses to external influences. On the other 
hand, the gene-theory, postulating factors in chromosomes, 
fits in well with the stable and change-resisting character 
of chromosomes. But in thus accentuating the relative 
non-mutability of the chromosomes as carriers of heredity, 
the most stable possession of the organism, the gene-theory 
sets off the process of heredity from differentiation of 
development; hence, as we have seen, it offers us no help 
in our attempt to explain differentiation. 

Now the gene-theory as formulated may not be the only 
way of interpreting the vast amount of reliable data accu- 
mulated by the numerous geneticists the world over. 
These many data, let us say at once, we accept. If, how- 
ever, we can offer another interpretation of them, can for 
example substitute for the factorial concept in the gene- 
theory another concept which is less rigid, more consonant 
with our knowledge of physiological processes and one 

1 Cf. Lillie, F. R., 192/; Conklin, 1924, et. al. 

2 Untutored savage man made his god as big as possible because 
hts god could do everything. It remained for the geneticists to 
make one of molecular size, the gene. Here obviously infinite 
minuteness means infinite capacity. According to one geneticist, 
Demerec, the gene has almost magic power. This is physico- 
chemical biology with a vengeance! 



substituting a means of protoplasmic reactions for a non- 
physical concept of molecules, we should be able to do what 
so far has been attempted in vain in biology, namely, to 
envisage differentiation and heredity as merely two expres- 
sions of development. 

It has been said that differentiation of development 
and genetics must remain forever separated. 1 And when 
and wherever geneticists have attempted a union of the 
two, they have failed. 2 But since heredity is expressed 
during the process of development and indeed is a kind of 
differentiation, biology must attempt to find ground com- 
mon to both. Up to now this common ground has not been 
located. I here propose a suggestion concerning the role 
of the gene in heredity and in differentiation. 

The moment that we postulate that differentiation dur- 
ing cleavage turns upon the taking up of potencies leaving 
free in the cytoplasm those that give the blastomeres their 
characters as such, we see that an organ becomes such 
because in the cytoplasm of its cells are those potencies free 
which make it a special organ. Every cell in an organism 
becomes what it is because its cytoplasm has free its par- 
ticular potencies whilst its nucleus binds all others. These 
latter would, if left unbound in the cytoplasm, act as 
obstacles to the display of the special potencies. Thus, 
the removal of potencies at the same time means removal 
of obstacles to cytoplasmic reactions. 

My fundamental thesis is that all the differences, i.e., 
differentiation, that appear during development, rest upon 
cytoplasmic reactions. These are made possible through 
removal of obstacles by nuclei, hence, by chromosomes and 
genes. The nuclei by removal of substances release the 
activity of the cytoplasm in one direction. The genes also 

1 Lillie, F. R., 192?, p. 36?. 

2 Morgan; Golds chmidt. 



act by removing impediments to cytoplasmic reactions. 
Let us examine this proposition more closely. 

I begin with the assumption that with the onset of the 
fertilizable condition of the egg there are in the cytoplasm 
all the potencies — chemical reactants — whence arise the 
future organs. Indeed, as experiments on fragments of 
eggs obtained during this period show, such cytoplasm has 
capacity to produce several embryos. The chromosomes 
in such an egg now begin to remove from the cytoplasm 
potencies so that others remain free to initiate reactions 
responsible for differentiation. Thus progressively restric- 
tion ensues. 

Contrast this condition with that in the history of the 
spermatozoon. With the two rapidly ensuing maturation- 
divisions the cytoplasm of the spermatocyte is divided 
among four spermatids giving rise to mature spermatozoa 
with less cytoplasm than the mature egg possesses. Egg 
and spermatozoon thus are markedly different with respect 
to the amount of their cytoplasm at the moment of fertiliza- 
tion. We may recall a further difference mentioned earlier 
in this book. Whilst eggs can develop in some cases with- 
out the spermatozoon, no spermatozoon has ever been 
found capable of development without the cytoplasm or 
at least a fragment of the cytoplasm of an egg. In 
capacity for development, thus, the egg is superior to the 
spermatozoon. This superiority is related to the egg's 
cytoplasm since this can develop either with egg- or sperm- 
nucleus. Now, according to our conception, the egg- 
nucleus gives up its potencies to its cytoplasm at some 
moment before the fertilizable stage. The sperm-nucleus, 
being likewise capable of taking up potencies again in the 
development of the egg which it fertilizes, must have lost 
its own potencies also before it takes up those residing in 
the egg-cytoplasm. When this loss occurs we do not know. 
A probable assumption is that as early as the moment when 



the sperm-cell as such is differentiated from all other cells 
and retains in its cytoplasm only the potency for sperm- 
ness, this conditions the nullification in some way of those 
potencies borne by the chromosomes. Or they may be 
lost during the process of the ripening of the sperm-cell, 
when, as is known, concomitant with morphological changes 
amino-acids are lost. A less likely assumption is that 
potencies of the sperm-nucleus are lost and nullified at 

In any case, both egg- and sperm-chromosomes are pre- 
pared for removing stuffs from the cytoplasm of the egg. 
I bring an example: If a spermatozoon from a Drosophila 
possessing pure red-eye fertilizes an egg of an also purely 
red-eyed female, in the cells which show redness, the egg- 
and sperm-genes remove from the cytoplasm the hindrance 
to the reaction leading to redness. The " factor/ 5 redness, 
is resident in the cytoplasm and expresses itself because the 
genes remove stuff opposing this reaction. If on the other 
hand the sperm-chromatin is descended from a white-eyed 
animal and the egg-chromatin from a red-eyed one, the 
sperm-chromatin, when in those cells which give the color to 
the eye removes stuff which releases whiteness-reaction, and 
the egg-chromatin stuff which releases redness-reaction, 
with the result that the cytoplasmic reaction is now no longer 
r + r = R as in the first case, but r + w — R{zv) where 
R{zv) gives either dominant red-eye color or an interme- 
diate color between red and white. Thus this conception, 
whilst consonant with the experimental findings of Men- 
delian genetics, differs from the theory of the gene for it 
places the determination of characters in the cytoplasmic 
reactions. The active "factors" for Mendelian characters 
do not reside in the genes; rather, the genes by extracting 
definite materials from the cytoplasm render possible the 
reaction of the cytoplasm-located hereditary factors. Only 
so far as they take out substance do the genes determine 



Thus finally every cell in the most complex organism 
has in its nucleus all the potencies bound except that one 
which is free in the cytoplasm and which makes the cell 
specific. The egg-cell, for example, the moment it becomes 
differentiated from other cells of the body is such because 
it has in its nucleus all bound potencies except that which 
makes it an egg-cell. And it is this potency in the cyto- 
plasm which determines the future growth and differentia- 
tion of the egg to that moment, when these bound potencies 
are thrown again into the cytoplasm. The sperm-cell is 
such because in its nucleus are all the potencies bound except 
that which determines the sperm-character of the cell. 
This potency determines the further differentiation of the 
spermatozoon and most probably is responsible for the 
nullification of the potencies in the sperm-nucleus. 

This conception of mine concerning the action of the gene 
offers a far better interpretation for several phenomena 
than the hitherto proffered conceptions. Thus, for exam- 
ple, can we state that sex-differentiation is resident in the 
cytoplasm. For the majority of animals, the taking out 
from the cytoplasm of more sexual potencies by the chro- 
mosomes results in femaleness. So in those experimental 
conditions, where more than one set of chromosomes is 
present, the organism becomes female. The taking out of 
fewer sex-potencies by the chromosomes gives rise to a 
male. Thus an egg with half the somatic number of chro- 
mosomes, the so-called haploid number, is always, so far 
as we know, a male. Finally, hermaphroditism means 
equal abstraction of sex-potencies by the chromosomes, 
leaving in the cytoplasm both male- and female-factors. 

These considerations warrant the assumption that the 
genes play a part in heredity but are not the factors of 
heredity in the sense of the geneticist; the genes act through 
their binding of potencies in such wise as to free the action 
of the cytoplasm-located factors of heredity — that is, 



chemical reactions in the cytoplasm underlie both differen- 
tiation and heredity. 

In thus relating heredity and the action of the genes to 
reactions in the cytoplasm, we come to a more physio- 
logical conception for heredity than the theory of the gene 
and one which instead of running counter to the facts of 
differentiation of development is consonant with them. 
From fertilized egg to the least active cell in the adult 
organism, the visible manifestations of life are outside the 
nucleus. It is the cytoplasm therefore that we consider to 
be the field of activities be they concerned with heredity 
or differentiation. 

With reactions in the cytoplasm deals the fifth event dur- 
ing cleavage enumerated above with which our discussion 
of the cause of differentiation is concerned: the redistribu- 
tion of water during cleavage. 

From fertilization through cleavage eggs of marine 
animals certainly and all others probably show a visible 
altered distribution of water. This is best observed in 
experimentally treated eggs, but it can also be seen in 
normal development, as I stated in the chapter on water. 
After the initial ectoplasmic dehydration which occurs when 
eggs are fertilized or subjected to experimental means that 
initiate development, the egg establishes a new equilibrium 
with the sea-water. On this level, water, as discrete drops, 
moves from place to place within the egg or from egg to 
external medium. The formation of water-drops is a 
rhythmical phenomenon which accompanies each division- 
cycle of the cleavage-period. 

The addition or removal of water in a reversible reaction 
determines hydrolysis or synthesis, respectively. Thus, 
water plays a role as a component in chemical reactions; 
in addition, it is both a solvent for other components and 
a part of the cytoplasmic structure, the chamber of the 
reactions. Then the demonstration that water is inter- 


mittently present as discrete drops in viable cells having 
had any one of several kinds of treatment — whose action 
is not deleterious since one can prove that the cell after 
treatment returns completely to the untreated state — has 
far-reaching significance: it becomes an index to the nature 
and rate of reactions. In both normal Qgg and blastomeres, 
the rhythmical appearance of water-drops allows us to 
correlate their occurrence with the rhythm in a cleavage. 
Differences in the formation of the water-drops in different 
blastomeres we may regard as an index to chemical changes 
underlying the progressive restriction that runs with 

With each succeeding cleavage the egg is further sub- 
divided into chambers of capillary dimensions. At the 
end of the cleavage-period the total surface-area of the 
blastomeres is much greater than that of the uncleaved egg. 
Thus, during cleavage the ectoplasm increases in amount. 
This increase of ectoplasm constitutes the last of the six 
enumerated changes that occur during cleavage. The 
cleavage-period may be defined as one in which the cyto- 
plasm diminishes through its conversion on the one side 
into new nuclear substance and on the other into new cell- 
partitions. Having examined the role of nuclear growth 
in differentiation, I seek now to evaluate the evidence 
from which I derive a postulate concerning the role of that 
part of the ground-substance located at the cell-surface, 
the ectoplasm, in differentiation. 

In the first line stands the fact that the ectoplasm 
increases in amount with the progressive sundering of the 
egg into blastomeres, regardless of the type of cleavage by 
which this result is reached. Whilst no animal egg is a 
perfect sphere and never by cleavage gives rise to blasto- 
meres which are perfect spheres, nevertheless the ratio of 
total surface to total mass which obtains for a sphere when 
sub-divided into spheres is closely approximated by the egg 



during cleavage; the mass of protoplasm does not increase 
but the total surface-area does. Increased surface-area 
is best seen in an egg with total cleavage which develops 
into a blastula. It is clearly shown by the egg of the star- 
fish for during early cleavage the blastomeres are at first 
separated from each other. In totally cleaving eggs which 
give rise to morulae, the increased surface area appears in 
the covering blastomeres; they are more flattened and the 
ratio of surface to mass thus is greater. For eggs with 
discoidal cleavage, it has been established that new cells 
are added to the embryonic disc from the underlying yolk 
where nuclei undergo mitotic division without being par- 
titioned off into cells; only when around those nuclei lying 
next to the disc cell-boundaries form, are new cells added 
to the growing embryonic disc. Cleavage-planes arise in 
superficially cleaving eggs only after the nuclei reach the 
ectoplasm. The difference between totally and partially 
cleaving eggs lies in the fact that the initial cleavages in 
the latter are confined to an ectoplasmic region which is 
utilized in the formation of cell-walls before additional 
ectoplasm forms; in the former, cleavage and increase in 
ectoplasm run more synchronously. 

Since the ectoplasm is a part of the living system and 
since during cleavage no living substance is added to the 
egg from the outside world, the source of new ectoplasm is 
from the egg-substance itself: ground-substance alone 
constitutes this source. 

But the interpretation of the role of the ectoplasm in 
differentiation does not rest only upon its amount and its 
source; in some of the events named as occurring during 
the cleavage-period, the ectoplasm plays a part. 

In the first place, consider the fact that a fertilized egg, 
which possesses pluripotency, develops normally only into 
one embryo. This means that only if the ectoplasm of the 
blastomeres is removed from its normal contact with the 



other blastomeres — as happens in the experiment of sep- 
arating the blastomeres — do they develop separately. In 
the intact cleaving egg the ectoplasm interacts with that 
of the neighboring blastomeres and thus insures the normal 
development into one embryo. 

Secondly, in normal development the egg cleaves into 
blastomeres, that is, new cell-partitions arise. When exper- 
imentally eggs are induced to differentiate without cleavage, 
abnormal development results. In such cases of differ- 
entiation without cleavage there is always an abnormal 
accumulation of ectoplasm. 

In the third place: the embryonic axis in eggs of radiate 
animals and the plane of bilateral symmetry of the 
embryo in eggs which develop into bilateral animals, arise 
in the surface and not within the bulk of the egg-substance. 
If there be gradients of development these are ectoplasmic 
and not axial, polar or otherwise. 

Fourthly, the ordering and shifting of cytoplasmic 
inclusions, in terms of primary causes, depend upon the 
ectoplasm, since the changes in the ground-substance, 
responsible for these movements, are themselves dependent 
upon activity in the superficial cytoplasm, as was shown 
in the chapter on cell-division. 

The importance of the ectoplasm as a causal factor in 
differentiation is in .addition predicated upon its general 
properties, its role in the exchange of water between egg 
and outside world, its necessity for fertilization, its signif- 
icance in parthenogenesis, its part in initiating division of 
the cell. These attributes alone would suffice to establish 
the ectoplasm as a leading factor in differentiation. But 
there are other and more general considerations. 

The establishment of new surfaces during cleavage alters 
the physical properties of the protoplasm. With the 
separation of the egg-substance into blastomeres the origi- 
nal physical state of the uncleaved egg is altered since some 



of it goes to make up new nuclei and another portion forms 
the new cell-boundaries. Thus, the viscosity of the cyto- 
plasm, for example, in the individual blastomere can not be 
the same as that of the cytoplasm of the uncleaved egg. 
Furthermore, the differential distribution of the cytoplas- 
mic inclusions during cleavage certainly alters the original 
physical state of the egg. 

With subdivision of the egg into blastomeres, changes in 
the chemical reactions take place. Each blastomere repre- 
sents a separate reaction-chamber of capillary dimensions 
favoring especially those reactions which are confined to 

Cleavage means a change in the space-relations of the 
original egg-substance. With the formation of new cell- 
partitions, especially in totally cleaving eggs which form 
blastulae, the subdivision into cells means new disposition 
of materials which at first were in more intimate contact. 
The orderly arrangement of blastomeres may be looked 
upon as one of the most characteristic attributes of the 
cleavage-period. The cell-surfaces are not simple ones 
but are made up of prolongations of unequal distribution, 
length, and activity. 

With cleavage, then, by virtue of the ectoplasm, the 
original egg-substance is separated into blastomeres and 
the blastomeres are integrated by means of intercellular 
connections, that is, the ectoplasmic prolongations. It is 
well known that cells in a strip of tissue, like a strand of 
ciliated epithelium, behave differently when part of the strip 
and when isolated. The beating of the cilia can be observed 
in an intact strip to run in order from one cell to the next; 
whereas when the cells are isolated the cilia beat irregu- 
larly. The same phenomenon has been observed in sus- 
pensions of spermatozoa. 1 In a normal sperm-suspension 

1 Lillie, F. R., 1913. 



each spermatozoon moves by the lashing of its tail. If the 
spermatozoa are caused to form balls by the addition of 
some substance which agglutinates them with their heads 
sticking together, the tails now beat one after the other 
in succession. It is also well known that cells of a tissue 
when isolated and grown in tissue-culture manifest form- 
changes and behavior which never occur when they are 
part of the tissue. 1 Finally, blastomeres when isolated 
reveal behavior arising out of their isolation. In brief, 
therefore, the behavior of blastomeres during cleavage 
must be in part due to an integration brought about by 
the intercellular connections arising from the ectoplasm. 

The movement of blastomeres is an undoubted factor in 
development and this movement is a function of the ecto- 
plasm. The rise of cells out of which is formed an embry- 
onic area in a specific region is to be attributed to a 
movement on the part of the cells which is closely akin to 
amoeboid movement. True, physical factors may be con- 
cerned in the displacement and new alignment of blasto- 
meres, but these are to be regarded as subsidiary. No 
purely physical theory has as yet accounted for the process 
of invagination by which a hollow blastula is converted into 
a gastrula. On the assumption, however, that cells take 
up a certain position with reference to others through their 
active movement we may approach the explanation of 
many processes, as invagination and evagination, epiboly, 

Finally, there is clear experimental evidence that during 
cleavage the ectoplasm reveals structural changes and 
activities which parallel cleavage-rhythms and which indi- 
cate that the ectoplasm plays a role in determining the 
direction of differentiation. Some simple observations of 
my own may be cited. 2 

1 Harrison, Lewis and Lewis, et al. 

- Just, I 9 2SC. 



If eggs of Arbacia be exposed to hypotonic sea-water dur- 
ing the time after fertilization when the vitelline membrane 
is being separated and are then returned to normal sea- 
water, they develop throughout the cleavage-period much 
as untreated fertilized eggs. A sharp difference is noted, 
however, in the blastula stage, for then, whereas the normal 
blastula is made up of cylindrical cells enclosing a cavity, 
the eggs which have had treatment with hypotonic sea- 
water are made up of cells approaching the cuboidal form; 
the cilia of their cells are longer than those on the normal 

If the eggs of the same species of sea-urchin are treated 
three minutes after fertilization, that is, two minutes later 
than those used in the observation described above, part 
of the egg protrudes beyond the membrane and these eggs 
develop as twin embryos. In some cases the twins remain 
joined, in others they become separated. I have found by 
exposing eggs from the same females at different intervals 
of fifteen or thirty seconds after fertilization that the most 
favorable time for the production of these twins comes 
immediately following the separation of the membrane 
from the entire egg. Thus, the same treatment if given in 
a different stage of the egg's development, yields a different 
result. This statement holds generally for the different 
periods during the cleavage-process; to treatment with 
hypotonic sea-water, eggs in different stages of development 
respond differently as shown by the difference in the types 
of embryos resulting. 

Now the variety of the results obtained in these observa- 
tions can be related to ectoplasmic behavior. Beginning 
with the moment of fertilization the ectoplasm goes through 
changes in behavior which are easily followed under the 
microscope. One needs, therefore, only to treat the egg 
during periods when by observation one notes a difference 
in the quality of ectoplasmic behavior in order to obtain an 
alteration in the development. 



The evidence that the treatment affects the ectoplasm 
alone is derived from the following considerations: First, 
there are no other noticeable or as striking changes elsewhere 
in the egg. For instance, the time for obtaining blastulae 
with cuboidal blastomeres and long cilia comes when the egg- 
surface is breaking down in the process of separating its 
vitelline membrane. On the other hand, the best time for 
the production of twins comes when the ectoplasmic surface 
is being reconstituted, or, one might say, a new surface 
arises. Now at these moments, separated by a time inter- 
val of about two minutes, no profound changes either in 
the egg- or sperm-nucleus or in the cytoplasm below the 
ectoplasm can be noted. Secondly, the method employed 
in these simple observations is that of treating the eggs 
experimentally for fifteen or at the most thirty seconds. 
An exposure to hypotonic sea-water of this duration is 
most certainly to be regarded as affecting the egg-surface 
only. I conclude, therefore, that the various alterations 
in the development called forth by treating eggs with 
hypotonic sea-water at intervals after fertilization are due 
to an effect on the ectoplasm. As development ensues, 
the ectoplasm undergoes definite changes correlated with 
the definite periods in development. If these surface 
reactions are modified, development is modified. I may 
refer again to the phenomenon of differentiation without 
cleavage. Certainly this represents an extreme type of 
developmental modification. As certainly it indicates 
that the alteration is due to modification in the behavior of 
the egg-surface. 

Finally I cite an observation on the egg of Asterias 1 . 
Normally during early cleavage the blastomeres of this 
egg lie within the vitelline membrane apart from each 
other; later, regaining contact they develop into one 
embryo. When after the second cleavage the four blasto- 

1 Jusu 1931. 



meres lie apart, they may with care by puncture of the 
vitelline membrane be removed as four independent cells. 
If brought together again and kept in close contact, by 
surrounding them with fibres of lens paper, they unite and 
develop into a single embryo. Also, two blastomeres from 
one egg when brought together with two from another in 
some cases united and developed into one embryo; often 
however union failed to take place. I found that this 
failure resulted whenever the transferred blastomeres were 
not in exactly the same moment of development. For 
example: two lots of eggs from the same female were fer- 
tilized at an interval of one minute apart from each other. 
At second cleavage, the four blastomeres were removed 
from one egg of each fertilized lot. Two blastomeres of the 
one were next brought together with two blastomeres of 
the other; no union was established. Observation revealed 
that the surface-changes in the transposed blastomeres 
were different. This observation furnished the clue for 
the failure of union between transposed blastomeres taken 
from a lot of eggs which had been fertilized at the same time : 
For the establishment of the interconnection of blastomeres, 
the ectoplasmic condition must be identical. Therefore, the 
union of blastomeres can be made certain if by direct obser- 
vation their surface changes have been found to be in the 
same stage. Since in any lot of eggs in best condition fer- 
tilization takes place in every egg at almost the same instant 
and the development ensues at much the same rate, at 
second cleavage the difference in the ectoplasmic behavior 
between the most slowly and the most rapidly developing 
egg is never as great as that in eggs fertilized at an interval 
of one minute. The failure of blastomeres from two eggs 
of the same fertilized lot to unite therefore is to be attrib- 
uted to changes in ectoplasmic behavior resulting from a 
difference in rate of development which is of seconds only. 
To mv knowledge, there exists no observation which so 



clearly and so beautifully shows the quickly occurring 
changes in ectoplasmic activity. 

The ectoplasm stands not simply as a barrier of the cell 
against the outside world; it is also the medium of exchange 
between cytoplasm and environment. As such, it is the 
first cell-region to receive impressions from the outside 
world; through its delicacy of adjustment and fineness of 
reaction, it constitutes the first link in the chain of cyto- 
plasmic reactions and sets the path for the orderly suc- 
cession of events comprising the course in the differentiation 
of development. 

The moment to moment changes in the ectoplasm in 
its response to the cell's milieu set up conditions in the 
cytoplasm which are favorable to the mechanism of the 
synthesis of nuclein, i.e., of the building up of nuclei out 
of cytoplasm. This synthetic reaction constitutes the 
removal of a barrier to those cytoplasmic reactions leading 
to differentiation. Alongside this function of setting up 
conditions favorable to the releasing-reaction of nuclear 
syntheses, is the growth of ectoplasm through the transport 
of ground-substance to the cell-surface. The ectoplasm 
impresses the cytoplasm, and this ectoplasm-induced cyto- 
plasmic activity brings about the nuclear behavior which 
underlies genetic restriction. Ectoplasmic behavior is thus 
the direct and so far the only visible manifestation of the 
cause of the differentiation of development which takes 
place during cleavage. 



Chromosomes and Ectoplasm 

Jl$ a large measure our knowledge of the effects 
of experimental means on cells is the result of studies on 
eggs. But in these studies the primary effect of the experi- 
mental means on the protoplasmic system was often mis- 
interpreted. Enthralled by the dramatic manoeuvres of 
the chromosomes in mitosis investigators assumed for the 
chromosomes an independence from the remainder of the 
cell; this point of view easily led to the assumption that 
external agents directly affect the chromosomes. So also, 
the origin of mutations has been predicated upon inherent 
activity of the chromosomes. 

The effects on marine eggs of changes in temperature, in 
salinity or in hydrogen-ion concentration vary, depending 
upon the eggs employed. For a species of eggs these effects 
vary before and after insemination. For the fertilized egg 
again there is a differential effect of the means which runs 
with the mitotic cycle. But there is here no evidence to 
indicate that this effect is primarily or only on the nucleus 
or its chromosomes; what the experiments do show is that 
the effect is first on the ectoplasm. 1 

These changes in the surrounding medium bring about 
changes in the nucleus only secondarily. Always the ecto- 
plasmic changes come first. Indeed, within optimum 
range, the magnitude of the ectoplasmic changes determines 
that of the nuclear. There is also a relationship between 
the duration of the exposure of the egg to these changed 

1 Just, 1932. 



environmental factors and the quality and character of 
the ectoplasmic reaction. Again the intensity, that is, the 
strength of the change in the environment, determines the 
rate of the ectoplasmic response; and within optimum 
range, this rate determines the degree of the nuclear 

Specific examples to support these statements one may 
find in the literature on fertilization, experimental par- 
thenogenesis, experiments on cell-division and on develop- 
ment. I may mention again the observations on the egg 
of Chaetopterns which after treatment with hypertonic sea- 
water develops and differentiates into an abnormal swim- 
ming form; here clearly ectoplasmic activity conditions 
nuclear behavior. In sea-urchins' eggs treated with potas- 
sium cyanide, the failure of mitosis runs with an exagger- 
ated activity of the ectoplasm. 1 Also, in sea-urchins' 
eggs, the experimental prolongation of the monaster 
stage is conditioned by an exaggerated ectoplasmic 

Egg cells have been subjected not only to changes in 
temperature, in salinity and in hydrogen-ion concentration 
of the surrounding sea-water; they have also been exposed 
to radium, Roentgen and ultra-violet rays. The first 
noticeable effect of such exposures is on the ectoplasm. 

Some years ago Packard 2 reported results of exposing 
eggs of Nereis to radium. The first effect observed is 
cytoplasmic. In consequence or at least in sequence to 
this follow modifications in nuclear behavior. Redfield 3 
especially has analyzed the ectoplasmic response displayed 
by this egg after exposure to various rays. In this same egg 
I found that ultra-violet rays induce profound local ecto- 

1 Just, 1927b. 

2 Packard, 1914. 

3 Redfield, ip/S. 



Fig. 37. — Egg of Nereis with first maturation spindle held near the centre of the 
cell as an effect of ultra-violet irradiation. 



■ ■.■','■•> V 

••:•■••,: /.- 

Fig. 38.- Irradiated egg of Nereis showing nuclear division without extrusion of 

polar bodies. 



plasmic changes which are followed by abnormal nuclear 
behavior. 1 

The effect of the rays is limited to the superficial cyto- 
plasm, whose altered condition brings about the remarkable 
result that the eggs develop with seventy chromosomes 
instead of the normal number, twenty-eight. The pro- 
cess is as follows : Consequent to the injury of the ectoplasm, 
the normal cytoplasmic movements are inhibited. As an 
effect of this inhibition the first maturation-spindle formed 

Fig. 39. — Irradiated egg of Nereis with Fig. 40. — First cleavage spin- 
four groups of chromosomes resulting die, irradiated egg of Nereis, 
from division of the two spindles in showing some of the seventy 
Fig. 38. chromosomes. 

after break-down of the germinal vesicle fails to move to 
the surface of the egg. Instead of at the periphery — to 
which normally the spindle moves and where after separa- 
tion of the chromosomes their outer group is extruded with 
the first polar body — the first maturation mitosis takes 
place at or near the centre of the egg (Fig. 37), two nuclei 
arise and from each of these a spindle forms, each giving 
rise to two groups of chromosomes (Fig. 38). In other 
words, the first and second maturation mitoses take place 
near the centre of the egg and four egg-nuclei arise (Fig. 
39). These four nuclei with the sperm-nucleus, each con- 
taining fourteen chromosomes, give rise in some cases to 

1 Just, 1926a, 1933b and c. 



a cleavage-spindle with seventy chromosomes (Fig. 40). 
In other cases multi-polar (two, three or more) spindles 




^ . r •"• . .■■•.:■ 

• * * * r 

Fig. 41.— A multipolar first cleavage spindle, irradiated egg of Nereis. 

.**■ *■ ' ; 

I V/jp 

v >i* *•♦:•> 

Fig. 42. — Another type of multipolar first cleavage spindle, irradiated egg of 


arise as the appended figures (Figs. 41 and 42) of the radi- 
ated Nereis egg show. 



The striking similarity between these figures of abnormal 
mitoses and those found in cells of human cancer deserves 
some comment. 

Almost fifty years ago Galeotti described and figured 
abnormal mitoses in human cancerous cells. Later he 
showed that poisons, acting on skin cells of the salamander, 
induce pathological mitoses closely resembling those found 
in cancer. Subsequently various investigators have con- 
firmed Galeotti's findings. Cancer cells of man and of 
other mammals in tissue culture likewise show abnormal 
mitoses. If one compares under the microscope my prepa- 
rations of these abnormal mitoses in eggs of Nereis with 
those of human cancer cells, one notes at once that, except 
for their greater clearness and sharpness due to more excel- 
lent technique, the mitotic figures in the eggs bear the 
closest resemblance to those in the cancer cell. How far 
this resemblance has common cause, it is hazardous to 
assert. And this for the following reasons. 

In the first place, eggs of a worm, freshly discharged 
into the sea, are far removed from the cells of warm blooded 
animals, which in the intact organism stand in a quite 
different relation to their environment, conditioned by 
nervous and humoral integrations. Conclusions for human 
cells derived from studies on cells of other mammals even 
are not always safe. Hence we are wary in making final 
statements on the basis of animal experimentation concern- 
ing human disorders, the more so since man varies so much 
one with the other, and since in one individual the har- 
monious adjustment of cells, their capacity for self-regula- 
tion, so greatly depends upon that so little understood 
principle of individuality, which in the final analysis goes 
back to the chemical make-up of the ground-substance of 
every single cell; 1 from it emanate what we designate indi- 
vidual resistance and susceptibility. 

1 J^t, 1936b. 



In the second place, the fact that abnormal mitoses 
arise in response to most diverse means needs to be con- 
sidered. The Nereis egg which portrays them as an effect 
of radiations, responds in like manner to other means. 1 
Then the reaction is an expression of independent irritability 
which reveals specific structure. So with somatic cells 
which react similarly. Thus, abnormal mitosis does not 
as such mean that the cell is one of cancer. Moreover, 
not every human cancer cell displays abnormal mitosis. 

None the less is the resemblance striking. Far removed 
though any cell — egg of worm or cell of an onion root tip- 
is from a human cell of cancer, it might not be too far fetched 
to see in the similarity of response a manifestation of a 
fundamental structure common to all protoplasmic systems. 
It is as imperative for the problem of cancer as for any 
problem in biology discussed in this book that we add to 
our knowledge of this structure. 

The fact that the egg of a worm and a human cell show 
comparable configuration in abnormal condition points to 
a similarity in a basic pattern of organization in living mat- 
ter upon which the characters of speciation are imposed. 
If therefore we could designate in the egg one characteristic 
deformation as diagnostic of its response to changes in the 
external medium, we should have at hand a point of depar- 
ture for study of the cancer cell. Now such diagnostic is, 
as I shall show, available: All means that elicit the rise of 
abnormal mitosis do so through alteration of the ectoplasm. 
A more exact cytology of human cancer cells with reference 
to ectoplasmic behavior is thus indicated. 

The effect of the radiation on the ectoplasm of the egg 
of Nereis is shown in the following ways. First, the normal 
egg after fertilization extrudes a jelly throughout the whole 
ectoplasm so that every fertilized egg is enclosed in a hull 

1 Just, 1936c. 



of jelly of the same consistency throughout and of equal 
width. The ectoplasm whence the jelly escaped shows no 
persistent localized inequality. The changes taking place 
in it are not confined to any particular region. The egg 
fertilized after radiation, on the other hand, extrudes jelly 
of lesser degree of consistency so that the eggs tend to fall 
out of their jelly hulls. Always is there present in the 
ectoplasm thereafter an injured region marked by the 
change in the structure of the ectoplasm as well as by 
the greater distance of the vitelline membrane at this site. 
This change in the physical make-up of the jelly is an index 
of the ectoplasmic injury induced by radiation. In the 
second place, in the radiated eggs the first cleavage plane 
passes through the mark of injury revealed after the extru- 
sion of the jelly. Third, a localized area of injury in the 
ectoplasm persists through the egg's development and can 
be traced into the larval worm. 

This observation on the effect of ultra-violet rays on 
the Nereis egg is supported by my findings on the egg of 
Chaetopterus; in a similar, but perhaps not so striking a 
manner, ultra-violet rays affect the surface cytoplasm of 
this egg. 1 

Since in some cases radiations, especially radium and 
Roentgen rays, are most effective after nuclear breakdown, 
it is often held that irradiation does not affect the cell while 
its nucleus is intact. From this it is argued that radiation 
directly affects the chromosomes. Indeed, some workers 
consider that the action of Roentgen rays, for example, is 
limited to the chromosomes alone. Against this position 
arguments may be adduced. 

For many cells it has been learned that the display of 
normal mitotic activity depends upon some condition in 
the cytoplasm which often may even be visible. It is well 

1 Just, i9Jod, 1934c. 

347 ^/d^^^-r 


known that a certain complex of physical and chemical 
conditions in the cytoplasm is necessary for the initiation 
and completion of mitosis. The rhythm of nuclear division 
is itself parallel with the rhythm of structural and physico- 
chemical changes in the cytoplasm. Abundant evidence 
indicates that parallelling the rhythm of mitosis is a rhythm 
of susceptibility and resistance of the cytoplasm to many 
and diverse experimental means. In some cases, notably 
that of the egg of Nereis, the experimental means Including 
radiations are as effective on the egg in the condition of the 
resting nucleus as in stages of mitosis. The susceptibility 
displayed by a nucleus is to be correlated with changes 
in the cytoplasm; and since radiations are not alone among 
means which may be more effective on cells in stages of 
mitosis than on those with the so-called resting nuclei, we 
can not denominate Roentgen or radium rays as specific 
chromosome-affecting means. 

What is true is rather this: radiations are far nicer means 
than, for example, hypotonic and hypertonic solutions, 
heat and cold — all of which give effects similar to those of 
radiations. Radiations produce effects which are more 
rapid, more exactly measurable and more widespread in a 
given population of cells. This, in my judgment, is the 
chief value of radiations as experimental means. 1 Failure 
to appreciate that in experiments with radiations the cyto- 
plasm is affected and that radiations belong to a large class 
of experimental means has given birth to the fertile error 
that Roentgen, radium and other rays are causative factors 
for inducing directly profound changes in chromosomes. 
The offsprings of this notion, nursed by the gene-theory 
of heredity, are theories concerning the origin not 
only of species but also of the whole realm of living 

1 Just, i93 6 c- 



The fact that in Roentgen therapy there is a difference 
in the susceptibility of human cells does not vitiate the 
argument. Among these cells those most highly endowed 
with division and growth capacity are most susceptible — a 
fact which does not prove that the rays are chromosome- 
specific in their action. Rather, because of such division- 
and growth-capacity, the cells have cytoplasm whose con- 
dition renders them more susceptible to radiation than 
other cells. 

As we have seen, the effect of the feebly penetrating 
ultra-violet rays is a sharply localized ectoplasmic injury. 
Hence, it is not the penetrating power of the rays and so 
their power to reach the more deeply lying chromosomes 
which is responsible for their effects. Even the more deeply 
penetrating rays, Roentgen and radium, as Redfield has 
shown, also affect the ectoplasm; we therefore can not 
assume an effect of these radiations that is limited to chro- 
mosomes only. 

Further, materials entering the cell — gases, water, etc. — 
come into relationship first not with the nucleus but with 
the ectoplasm. We may here reason from analogy. Take 
oxygen consumption, for example. 

There was a time when biologists assumed the nucleus 
to be the seat of cellular oxidations. The presence of iron 
in the nucleus was postulated as part of the oxidation 
mechanism. On a priori grounds one would assume that 
oxygen entering the cell would combine with cellular constit- 
uents lying in the cytoplasm between the cell boundary 
and the nucleus. Now we know that this is at least nearer 
the truth: oxygen consumption is a function of cytoplasmic 
structure. Moreover, the attempt to demonstrate the 
presence of iron in the nucleus of spermatozoa failed though 
impeccable methods were used. 1 What is true of oxygen is 

1 Cf. Lynch, 1922. 



doubtless true of other substances that enter cells: they 
react first with the cytoplasm. 

Experimental cell-study gives instances of aberrant 
behavior of the chromosomes in egg cells. All of this work 
indicates that the experimental means first affects the 
ectoplasm. According to the theory here advanced this 
ectoplasmic effect is responsible for secondary effects on 
the whole egg cell. Among these is the aberrant behavior 
of the chromosomes. 

In the early days of the modern work on genetics, the 
experimental analysis could not have got very far without 
the descriptive studies on the behavior of the chromosomes. 
In the first line of this classic period for cytology stands 
Boveri's work. According to his analysis of experiments 
on dispermic fertilization, normal development depends not 
on the number but on the proper combination of chromo- 
somes. These experiments furnished part of the basis for 
the theory of the individuality of chromosomes. 

To secure dispermic or polyspermic fertilization of an 
echinid egg, one must first weaken its ectoplasm or employ 
heavy insemination. Where polyspermy ensues without 
experimental weakening of the eggs, we must assume that 
they were weak at the outset. The aberrant development 
of the blastomeres in his experiments Boveri related to the 
wrong combinations of chromosomes. But these combina- 
tions themselves depend upon the weakened conditions 
in the cytoplasm which make dispermy possible. In the 
normal egg the unimpaired ectoplasm protects against dis- 
order of the chromosomes. 

Aberrant behavior of paternal chromosomes is revealed 
in cross-fertilized echinid eggs when in certain crosses the 
paternal chromosomes, failing to take part in the mitotic 
process, are eliminated from the spindle. The possibility 
for cross-fertilization in all these cases is rendered greater 
by injuring the ectoplasm of the eggs. Here again, there- 



fore, the condition of the cytoplasm determines the behavior 
of the chromosomes. To induce cross-fertilization espe- 
cially between widely separated species, for most eggs at 
least, one must impair the integrity of the eggs' ectoplasm. 
Such impairment means a weakened cytoplasm. As with 
the aberrant behavior of the chromosomes in experimental 
polyspermy, so with that in cross-fertilization: it follows 
injury to the egg's ectoplasm. 

A point here must be emphasized. Too frequently bio- 
logists speak of the incompatibility of chromosomes to 
account for the elimination of chromosomes in cross-fer- 
tilization. As a matter of fact, chromatin may be elimi- 
nated in straight fertilized eggs. I have found in straight 
fertilized eggs of Echinarachniits that the whole egg-nucleus 
may fail to take part in the cleavage-mitosis. 1 Such eggs 
are injured. 2 Here there can be no question of the "incom- 
patibility" of chromosomes to foreign cytoplasm. Rather, 
the chromosomes fail to take part in the ensuing mitoses 
because of the weakened condition of the cytoplasm pre- 
viously treated. Work on the effect of temperature and 
of ether on the sperm-nucleus in the echinid egg are ame- 
nable to the same interpretation. The behavior of monaster 
eggs is also a case in point: the abnormal behavior of chro- 
mosomes is clearly due to injury of the superficial cytoplasm 
brought about by vigorous shaking at a time after insemina- 
tion when the ectoplasm is very susceptible to experimental 
treatment. The effect of radiation referred to above on 
straight fertilized eggs may be recalled. Here again the 
aberrant behavior of the chromosomes is in consequence 
of a definite ectoplasmic injury. Finally, Dubois has shown 
for the egg of Sciara that chromosomes are normally elimi- 

1 Just, 1924. 

2 See also J. Gray on Echinus-egg. 



nated during cleavage and that this elimination is deter- 
mined by the ectoplasm. 1 

In all the foregoing it is safe to conclude that the injury 
to the cytoplasm is an ectoplasmic injury. We may there- 
fore proffer the hypothesis that in ectoplasmic behavior 
lies the cause of the behavior of the chromosomes. Normal 
chromosome-distribution and -combinations depend upon 
the integrity of the ectoplasm; their aberrant behavior is 
the effect of the loss of this integrity. Such behavior may 
manifest itself in chromosomal elimination, fragmentation, 
and the like. 

To me the conclusion seems inescapable: ectoplasmic 
behavior determines the cytoplasmic reactions that lie at 
the basis of nuclear activity in both normal and abnormal 
mitoses. Where experimentally induced mutations are 
related to visible changes in the chromosomes, as their 
fragmentation, translocation, etc., these are not to be 
regarded as direct effects of the experimental means 
employed, — e.g., temperature, radiations — but as express- 
ions of the antecedant altered cytoplasmic reactions brought 
about by changes in the ectoplasm that evolved in response 
to the action of the means. 

If one observes under a microscope, equipped with a 
dark-field condensor, a suspension of Chinese ink ground 
up in water, one sees these inanimate particles scintillating 
back and forth in Brownian movement. One knows that 
the shining particles are moved and are not themselves 
motile. It is a far cry from the dance of ink-particles to 
the orderly movements of chromosomes in a living cell. 
And yet, it is warrantable to assume that as we look upon 
the shifting of chromosomes in cells, their constant distribu- 
tion during cell-division, generation after generation, we 
behold, not a motility inherent in these bodies themselves 

1 Dubois, i $33. 



but a behavior which expresses a force in the cytoplasm 
enclosing them. In normal mitosis, in experimentally 
induced mutations, the chromosomes express the underlying 
behavior of the medium in which they lie. They mark for 
us the ebb and flood of the cytoplasmic tides. These in 
turn are under control of ectoplasmic activity. 


l 3 

Ectoplasm and Evolution 

The principle of evolution is as firmly established 
as any in biology. The evolution-theory constitutes a 
fundamental postulate of the science of biology and has 
proved a guiding principle of uncalculable value for bio- 
logical research. Among biologists exists the almost unani- 
mous verdict that evolution took place. According to 
the prevailing opinion, the world of living things was 
evolved from a unicellular organism. 

It should however be emphasized that this first form of 
life was not that of some now existing protozoon. The 
word, Protozoa, literally means first animals; but we should 
bear in mind that among the Protozoa themselves evolution 
has taken place. We therefore assume that the first form 
of life was a simpler unicellular structure than the proto- 
zoan. From this both the Protozoa as we now know them 
and multicellular animals came, the former evolving in one 
direction, the latter in another. 

We encounter two questions: How did this first living 
thing arise ? What was (and is) the cause of evolution ? 

With the first question most biologists will not concern 
themselves — they deem it unanswerable and thus only 
provocative of fruitless speculation. And yet such specula- 
tion will always be alluring. The drama of the universe in 
the act now before us is a tremendously moving spectacle, 
but the prologue to its pageantry is also capable of moving 
the dullest imagination. One need, therefore, make no 
apology in voicing a note concerning the origin of the world 
of living things. The answers to the second question are 



innumerable, as is attested by the many and various theories 
of evolution. Those biologists who, after having weighed 
the evidence, accept the theory that animals and plants 
exist as products of evolution and reject the theory of a 
special creation for every living being that ever was and now 
is, by no means agree concerning the cause of evolution. 

To take up our first question: How did the first living 
thing arise? To put it otherwise: How out of non-living 
matter did life arise? 

The combination of chemical compounds from the envi- 
ronment to make up the first living thing must obviously 
have meant a separation from the environment, that is, 
the combination must have been peculiar, both physically 
and chemically; otherwise, there never could have come 
about its separation and the maintenance of its integrity 
apart from the environment. Now the moment that this 
peculiar combination of compounds arose, there must have 
begun reactions or responses of it to the environment — 
especially to temperature, to gases and to electrolytes. 
The chief characteristic of this original substance was its 
peculiar and complex organization, which set it apart 
from its environment; but at the same time it must have 
been responsive to environmental changes. Environmen- 
tal changes must in the first instance have brought about 
the combination of compounds peculiar to living substance, 
and in the second place must have conditioned its activity. 

This original mass of primitive protoplasm at first per- 
haps showed no high degree of differentiation, but we can 
scarcely imagine it as a homogeneous structure through- 
out; as such it could not have endured for any great length 
of time. The moment that we assume that a combination 
of chemical compounds was separated out from the envir- 
onment as a peculiar system, we must postulate some dif- 
ferentiation in the mass — which differentiation served to 
keep the combination of compounds intact. 



If, however, we assume that this combination of com- 
pounds separated from the environment was first purely 
homogeneous throughout, then there must soon have come 
a time when factors in the environment played upon this 
structure and so modified, if they did not determine, its 
behavior. It would be difficult to imagine a structure 
made up of the same elements or compounds found in the 
environment and maintaining its separateness from the 
environment without some structural difference from it. 

The moment that such a peculiar combination of com- 
pounds arose, it assumed life; it had response to its 
environment, both because it arose from it and had to exist 
in it. 1 

Living substance can not be considered abstracted either 
from time or from space. The organism can not be separated 
from its environment; they form together one inter-acting 
system. Two predominating characteristics exhibited by 
living organisms are: first, those changes which are time- 
ordered; and second, those which are environment-condi- 
tioned. Thus, the organism — a single cell, for example — 
changes from moment to moment and the rate of such 
changes becomes its differentiating characteristic; within 
the organism these changes tend to run in one direction — 
the building up of protoplasm from simpler compounds 
while life lasts. External to it the environment plays a 
part in yielding up the raw material for these changes 
and setting the conditions for the reactions in the living 
matter. In a certain sense we should not speak of the "fit- 

1 Although there is the possibility of life below that size that can 
be made visible by the microscope, zve must admit that we knozv little 
concerning the organization or structure of such ultramicroscopic 
organisms. Therefore, zve might most profitably begin with a 
hypothetical organism whose size is within the range of resolution 
by the microscope. Nevertheless, what is said of such a hypothetical 
structure might also hold for one of ultramicroscopic size. 



ness of the environment" or the " fitness of the organism"; 
rather, we should regard organism and environment as 
mutually adapted. 

The play of factors in the environment — of temperature, 
of gases and of electrolytes — upon the living organism 
must be first on the cytoplasmic surface. Even if we 
assume that the primordial living thing was a mass of 
homogeneous protoplasm structurally the same throughout, 
there must have early arisen a differentiation between sur- 
face and interior. 1 In the constant interchange between 
environment and organism reactions must have taken 
place first in the more superficially located cytoplasmic 
structure; these reactions would condition succeeding ones 
in the endoplasm. The first step in the evolutionary proc- 
ess, then, was a differentiation of the cytoplasm into ecto- 
plasm and endoplasm. The second step, according to 
this theory, was a nucleo-cytoplasmic differentiation. 

We have thus a picture of the primordial living thing as a 
mass composed of the prototype of the ground-substance 
in cells as we know them to-day which limited itself in 
space by a changed surface, its ectoplasm. In time this 
primordial thing showed a farther differentiation of a sub- 
stance which by opposing its more fixed character to the 
ever-changing mobile character of the ectoplasm tended to 
maintain the stability of this primitive protoplasm. Thus, 
nuclear substance arose. 

In thus postulating for the nucleus only a secondary 
origin I reject the theory that the first form of life was a 
chromatin-granule. 2 However attractive this latter theory 
may be to those who regard the ultra-filterable virus as 
living and to those who believe that the gene represents the 
fundamental living unit, 3 my speculations concerning evolu- 

1 Cf. Child igi$ on "Surface-interior patterns" 

2 Cf. Minchin, 1915* 

3 E.g., Jennings, 1936. 



tion derive their plausibility from their consistency with 
known facts of cellular phenomena: one of these indicates 
the elaboration of chromatin out of cytoplasm. More 
than once have I in the foregoing chapters referred to the 
origin and growth of chromatin out of the cytoplasm. On 
the other side, there exist no data to indicate that chromatin 
builds up cytoplasm. 

The same emphasis that I place upon the differentiation 
of ectoplasm out of the ground-substance in the genesis 
of the primordial living thing, I place upon it as a cause in 
the evolution of the animal and plant kingdom. 

Protozoa are classified into ascending orders on the basis 
of their ectoplasmic structure and behavior. Eggs develop 
into adults by virtue of ectoplasmic changes during differ- 
entiation. Among multicellular adult organisms, grades 
of complexity can be recognized: the more complex the 
organisms, the richer are their modes of integration as 
shown by comparative studies on nerve-systems. What 
makes man's brain the greatest among animals, is not the 
number of its nerve-cells — indeed, in a given area, the pri- 
mate brain has fewer cells than that of other mammals — 
but the richness of their connections. Other forms of inter- 
cellular integrations in multicellular organisms are also 

Let us recall once more the fundamental functions of 
living protoplasm. These are contraction, conduction, 
respiration and nutrition. The primordial contraction, let 
us say that exhibited by the egg or protozoan cell, involves 
the cell interior to a secondary degree only. The cilia of 
ciliated Protozoa are ectoplasmic structures. Muscle- 
contraction in the highest organisms is a phenomenon of 
the cell-surface. 1 

Also conduction, we found, is an ectoplasmic function. 
The transfer of the effect of a stimulus is ectoplasmic, as 

1 Hill et al. 



for instance in the fertilization of the egg. In tissues highly 
endowed with conductivity, as in those highly endowed 
with contractility, the predominating characteristic is rela- 
tively large surface area. In nerve cells, conduction travels 
over the nerve fibre (ectoplasmic prolongation), and is 
transferred by means of ectoplasm from one unit to another. 
By and large, it is reasonable to assume that both contrac- 
tility and conductivity are greater in surface-rich than in 
surface-poor protoplasm. 

The subsequent differentiation from a hypothetical first 
form of life into animal or plant we may suppose came about 
through the higher development of contraction and of con- 
duction by that form which evolved as animal. A deviation 
or less emphasis on these brought plants as such into being. 
It is surely on the side of the nervous tissue that animals 
and plants differ most. 

Cellular oxidation is a function of cytoplasmic structure. 
Again it is reasonable to assume that oxygen coming into 
the cell makes first some union with the superficial cyto- 
plasm, as we have seen. 

Now of three fundamental life-processes — contraction, 
conduction and respiration — respiration may be regarded 
as primary: on it depends all vital activity. Respiration, 
the same in both animals and plants, may have been largely 
responsible for the early separation between plant and 
animal. That is, those primordial individuals possessing 
most rapid rates of oxygen-consumption tended to oxidize 
themselves, or as cannibals, their like. Some, because of 
lesser intake of oxygen, tended to accumulate C0 2 and thus 
developed photosynthetic power as a means of protection. 
With this went also the building up from COo and water of 
carbohydrate polymers, including cellulose. 1 The presence 
of cellulose determined the disposition of the cytoplasm 
found in higher plants — that is, a cytoplasm of peripheral 

1 Cf. also Geddes. 



location enclosing a vacuome. Thus, respiration may 
have determined the difference between animal- and 

Animals evolved slowly or rapidly, farther and farther, 
depending upon the degree to which ectoplasmic behavior, 
its faculty for contraction and conduction, developed. The 
greater became these two forms of behavior, the more 
imperative became the need of exquisite means for respira- 
tory-exchange. Respiration, according to our assumption, 
played a part in establishing the difference between animal- 
and plant-nutrition. Animal nutrition advanced showing 
progressively increased utilization of large surface-areas for 
the play of reactions — in digestion and for absorption of 
the digested end-products. With this evolved in increasing 
complexity a circulating system which gained steadily in 
structural perfection whilst its correlation with the respira- 
tion-system increased ; and a more exact integration by 
nerves and finally by internal secretions arose. 

Thus all forms of behavior by which we recognize that 
a thing is alive express themselves in response to the envi- 
ronment in the activity either of the ectoplasm itself — 
as in unicellular organisms — or of structures which are rich 
in ectoplasm. The fineness and nicety of ectoplasmic 
organization increase progressively in the animal kingdom 
from the lowest to the highest organisms and thus parallel 
the course of evolution. This course, from the emergence 
of life out of non-life to the separation of animals from plants 
and farther to the unfolding of progressive complexity of 
animal-form, makes manifest the role and importance of 
the ectoplasm in evolution. 1 

This hypothesis of the evolution of the living world holds 
also for the more restricted problem of evolution, the 
origin of species. As I see it, the most valid criterion for 

1 Just, 1933a. 



determining that animals of a genus belong to the same 
species is the capacity of normal fertilization and the pro- 
duction of fertile offspring. As has been abundantly 
shown, fertilization of an egg by a foreign spermatozoon is 
only possible if the ectoplasm has been debased. The 
unimpaired ectoplasm is a barrier to fertilization by non- 
specific spermatozoa. Then species arose through changes 
in the structure and behavior of the ectoplasm. 

In the differentiation of ectoplasm from the ground-sub- 
stance we thus must seek the cause of evolution. 



C o?i elusion 


chapter, The Ectoplasm, can not be gainsaid. In all 
cells, of unicellular and multicellular animals, the existence 
of the ectoplasm can be demonstrated. We have seen what 
is its role in the phenomena of conduction, contraction, 
respiration, the intake and output of water — these all being 
general properties of all animal cells. More specifically for 
the animal egg, we have seen that without the ectoplasm, 
fertilization can not take place, that in both fertilization 
and parthenogenesis the response of the ectoplasm to the 
inciting means for development is prognostic for the quality 
of the future development; that in cell-division the ecto- 
plasm initiates the event by regulating the movements 
within the cytoplasm and that by redistribution of its 
structure and relocalization of its activity, it establishes 
new cell-surface; and that during differentiation the ecto- 
plasm increases in amount and reveals a differential activity. 
This behavior of the ectoplasm was shown to be one causa- 
tive factor in differentiation of development, the other 
being the building up of nuclear material out of the cyto- 
plasm. Thus the reactions underlying both differentiation 
and heredity were shown to be under the domination of 
cytoplasmic reactions, resulting from an interplay of both 
ectoplasm and nucleus with the cytoplasm. On this basis 
an interpretation of the action of the gene was offered. 
The behavior of the chromosomes themselves in normal 
cells and in experimentally induced mutations was shown 
to be dependent upon ectoplasmic activity. It was finally 



suggested that ecto-endoplasmic differentiation is a factor 
in evolution. 

Thus briefly the argument in favor of my theory of the 
ectoplasm. Let it be clearly understood, however, that 
the theory in no wise invalidates the conception of the 
protoplasmic system as the unit of the state of being alive. 
Rather, the whole argument upon which the theory rests 
aims at encompassing this conception in a fuller and more 
complete fashion than hitherto was possible. Because of 
the up to now existing failure to assign to ectoplasmic 
behavior a role in the integrative action of the living system, 
it has been necessary to emphasize what this role is. I 
wish very clearly again to state my position: life resides in 
the whole of the protoplasmic system taken as a unit — the 
phenomena of life are not to be dissociated from the integra- 
tion of the system's constituent regions, the integration 
which is the basic manifestation of the state of being alive. 

While life rests in the protoplasmic system as a unit, 
life is not static, is not structure only, but is the sum-total 
of activities; protoplasmic organization or integration, 
therefore, means orderly reactions as surely as it means 
space-distributions of the protoplasmic components. We 
may never come to know what life really is, but if we do 
approach such knowledge, it will be through appreciation 
of activities which constitute the protoplasmic integration; 
these we learn by their expression. Life is not confined to 
nucleus only and certainly not to constituent genes; nor 
yet does it reside in the ectoplasm alone. The significance 
of the ectoplasm is that it gives visible expression to these 

On the one side every living thing tends to fixity and 
rigidity, resistant to change. This tendency aids to pre- 
serve individual integrity which hands over intact from 
generation to generation the character of the organism. 
In the protoplasmic system the nucleus represents this 



conserving static Moment. On the other side, the living 
thing is highly mobile, changing with every change in the 
environment, accommodating itself and thus evincing 
capacity for self-regulation. In the protoplasmic system 
the ectoplasm is the region of active momentary changes in 
response to environmental conditions. 1 Within the proto- 
plasmic system is a stability that is derived from the inter- 
play of the more static and the more mobile factor. This 
stability thus brought about determines both that organiza- 
tion of matter called living thing and that specificity which 
sets off one living thing from another. 

To the varying states of the protoplasm due to the con- 
comitant and succeeding reactions we need especially to 
address ourselves. The region of the protoplasmic system 
in which these changes are most strongly revealed is the 
ectoplasm. Its most important characteristic is its change 
in time, its rapid response to outside conditions. No other 
cell-component exhibits this characteristic in a like degree. 

By re-evaluating structure and function of the proto- 
plasmic system, my theory of the ectoplasm has signifi- 
cance not only for the advance of biological investigation; 
it offers a point of view also for medicine — the highest 
form of applied biology — whence medical problems can be 

Medicine, as human biology, a welter of complex rela- 
tions, even less than the biology of other animals can depend 
upon the ultimate particles of physical science for the 
solution of its problems. 2 As protoplasmic systems, human 
cells are capable of study by comparable cytological tech- 
nique and experimental methods which used on eggs have 
furnished the particulars that give basis for my general- 
ization. An insistent demand for medicine today is, 
therefore, a far-flung attack on human cells as such. We 

1 Cf. Montgomery, 1904. 

2 See Sauerbruch, 1926, 1937. 



need more and more to supplement that histology of normal 
and pathological states which although it suffices for dia- 
gnosis nevertheless remains the study of tissues — and more 
often of organs — by most exhaustive study of the minute 
structure of every single type of human cell both in its 
normal and in its every available pathological state. 

The study in every type of cell of its ground-substance — 
i.e., of the protoplasm minus the inclusions and the products 
of metabolism — may win a new understanding of the func- 
tions of the liver, the kidney, the gut, the pancreas, etc. 
Extension of the descriptions of the ectoplasm in these 
various cells given by the older medical histologists may 
serve for a closer correlation of structure and function in 
health and disease. In spite of the view that cancer, for 
example, is caused by external agents and that purely micro- 
scopic investigations here are fruitless, nevertheless it may 
be a problem intrinsic to the cell. In oedema, forms of 
nephritis and all other pathological states in which the 
balance of water is disturbed, careful microscopic investi- 
gation aiming at the fullest possible description of the 
minutiae in the cell is called for. The white blood-cells used 
every day in the diagnosis of disease have not been suffi- 
ciently investigated beyond diagnostic needs; studies made 
by means of new methods of cell fixation and staining might 
yield results that would throw additional light on the 
changes both in number and in quality which white blood- 
cells show in various human diseases. In these three prob- 
lems of medicine are strong indications that the ectoplasm 
is involved. 

From consideration of the theory of the ectoplasm as a 
working principle in the attack of general problems in 
biology and its application to problems in medicine, let 
us turn to its philosophical implications. 

Biology being limited to protoplasmic organization and 
not being able to go below it without that life is destroyed, 



must establish its philosophy on this basis. Any philos- 
ophy of biology must take into account the activity of the 
superficial cytoplasm. 

The living thing is part of the natural world; it grows 
and lives on the stuff of which it is made and whence it 
came. Then living thing and outside world constitute one 
interdependent unity, as evolution teaches, as the develop- 
ment of an animal egg reveals. As the boundary, the living 
mobile limit of the cell, the ectoplasm, controls the integra- 
tion between the living cell and all else external to it. The 
ectoplasm is the means of exchange for incoming and out- 
going substances. It is keyed to the outside world as no 
other part of the cell. It stands guard over the peculiar 
form of the living substance, is buffer against the attacks 
of the surroundings and the means of communication with 

If we trace the development of the brain in higher ani- 
mals, including man, we find that always it arises from 
ectoderm cells, those cells that possess most ectoplasm. 
The whole nervous system arises in animals always on the 
outer surface of the embryo; only later in development does 
it become enclosed within the body by other cells. Then 
the functions of the brain represent highly developed general 
properties of primitive ectoplasm. The brain can not be 
fully appreciated unless we bear this in mind. As with 
brain, so with sense organs. 

The origin and development of the brain show that a 
conception which assumes that either the individual alone 
or only the outside world is real, has no biological basis. 
The interdependence between individual and outside world 
is a postulate which has its sanction not from any abstract 
philosophical principle, but is true because of the biological 
basis here set forth. The best system of philosophy, then, 
is that which recognizes living thing and outside world as 
one interdependent continuum. Instead of building our 

3 66 


philosophical theories of life on the behavior of electrons, 
it is safer to erect them on a biological basis. We conceive 
human behavior in terms of the history, the evolution, of 
the differentiation of the cytoplasm, as this differentiation 
appears in the course of development of the living world, 
attaining its highest degree in the human race and in the 
human individual. 

The activity of the brain means a manifestation of ecto- 
plasmic properties which we may regard as evolved from 
primitive ectoplasmic relation to outside world. Every 
mental state may thus be conceived as having behind it 
this old relation. Perhaps it is because of this that man is 
able to trace the evolution of the universe. Our minds 
encompass planetary movements, mark out geological eras, 
resolve matter into its constituent electrons, because our 
mentality is the transcendental expression of the age-old 
integration between ectoplasm and non-living world. 

Life is not only a struggle against the surroundings from 
which life came; it is also a co-operation with them. The 
Kropotkin theory 1 of mutual aid and co-operation may be 
a better explanation of the cause of evolution than the 
prevailing popular conception of Darwin's idea of the strug- 
gle for existence. The means of co-operation and adjust- 
ment is the ectoplasm. But we can go farther. 

Man with his highly complex nervous system constitutes 
a species apart from the rest of the animal kingdom. 
Nevertheless he maintains communion both with animate 
and with inanimate nature. Still closer is his relationship 
with fellow man. These relationships rest upon a purely 
biological principle. The foregoing pages have established 
this thesis. Here, then, is indicated where we may seek 
the roots of man's ethical behavior. 2 

1 See also Ch. IV of Darzvin's "Descent of Man." 

2 In a forthcoming essay , / deal with this point at greater length. 



Nature is both continuous and corpuscular. In the 
former sense, we pass from lower to higher revelations of 
organization almost insensibly and with scarcely a break. 
Every form of matter follows upon another. In the latter 
sense, we recognize breaks in natural states from electron 
to atom, from atom to molecule, from molecule to com- 
pound, and from compounds in association to living matter. 
But even conceived of as corpuscular, matter, as we know it, 
is never purely discrete and absolutely independent from 
the remainder of nature. Whether we study atoms or 
stars or that form of matter, known as living, always must 
we reckon with inter-relations. The universe, however 
much we fragment it, abstract it, ever retains its unity. 

The egg cell also is a universe. And if we could but 
know it we would feel in its minute confines the majesty 
and beauty which match the vast wonder of the world 
outside of us. In it march events that give us the story 
of all life from the first moment when somehow out of 
chaos came life and living. That first tremendous upheaval 
that gave this earth its present contour finds its counter- 
part in the breaking up of the surface of the egg which 
conditions all its life that is to follow. The sundering of 
the egg into many parts, to be woven again into a whole 
is no less wonderful than the breaking up of the primeval 
unit out of which the sun and the stars, the earth and the 
moon were made separate and brought together again in 
the pattern of the heavens and the earth. 

The lone watcher of the sky who in some distant high 
tower suddenly saw a new planet floating before his lens 
could not have been more enthralled than the first student 
who saw the spermatozoon preceded by a streaming bubble 
moving toward the egg-centre. And as every noviate in 
astronomy must thrill at his first glance into the world of 
stars, so does the student to-day who first beholds this 
microcosm, the egg-cell. For the student of Nature there 



is always that which moves him, that something which 
he can not reduce. And out of this intuition he comes to 
know what otherwise would remain hidden. 

We feel the beauty of Nature because we are part of 
Nature and because we know that however much in our 
separate domains we abstract from the unity of Nature, 
this unity remains. Although we may deal with particu- 
lars, we return finally to the whole pattern woven out of 
these. So in our study of the animal egg : though we resolve 
it into constituent parts the better to understand it, we 
hold it as an integrated thing, as a unified system: in it 
life resides and in its moving surface life manifests itself. 




Agassiz, A. 1874. Embryology of the ctenophorae. Mem. Am. Acad. Sci., X. 

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Index of Authors 

Agassiz, 91 

Allman, 2 

Altmann, 63 

Andrews, E. A., 93 

Andrews, G. F., 96, 103, 117, 141. 278, 

Appelof, 91 
Armstrong, 43 

v. Baer, 2S7 

Barcroft, 225 

Bataillon, 225 

Bataillon et Tchou, 225 

Bayliss, 140, 145 

Bensley and Gersh, 56 

Bergmann, 96, 97 

Bernard, CL, 225 

Berthold, 96, 117, 27S 

Bohm, 100 

Bonnevie, 203 

Boveri, 26, 180, 182. 237, 304 

Brachet, 181 

Bradley, 41 

Brauer, 208 

Briicke, 2 

Buchner, 212, 213 

Butschli, 63, 88, 93 

Calberla, 100 

Cerfontaine, 99, 167, 302 

Chambers, 264 

Child, 301, 357 

Chun, 91 

Ciamician, 91 

Coe, 93 

Conklin, 66. 91, 283, 306, 310, 325 

Delage, 34, 180, 186, 212. 
267, 290, 297, 322 

^45> -5i, 


Demerec, 325 
Derbes, 20, 104, 105 
Descartes, 34 
Doflein, S8 
Dubois, 351, 352 
Dujardin, 2 

Erlanger, 93, 268 
Ewart, 284 

Faure-Fremiet, 102, 280 
Fischer, A,, 65, 66 
Fischer, E. ? 5 
Flemming, 49, 77 
Fol, 91, 96, 105, 199 

Galeotti, 345 

Gatenby, 89 

Geddes, 146, 359 

Gerould, 96 

Goette, 43, 97, 101 268 

Goldschmidt, 326 

Goldschmidt u. Popoff, 279, 2S1 

Gray, 264, 279, 280, 281, 351 

Greef, 213 

Groom, 98 

Gutstein, 88 

Haeckel, 75, 88, 89 

Haecker, 180 

Halkin, 92 

Hammar, 96, 122, 277 

Hardy, 65, 66 

Hargitt, 91 

Harm, 91 

Hartridge, 1 19 

Harrison, 82, 83, 84, 85, 120, y^ 

Hatschek, 168, 169, 203 

Heberer, 180 



Heider, 98 

Heilbrunn, 44, 222 

Heisenberg, 13, 14 

Hempelmann, 97 

Henle, 76 

Herbst, 281 

Hercik, 271 

Herfort, 100 

Hertwig, 0., 96, 214, 215 

Hertwig, 0. and R., 117, 203 

Hibbard, 57 

Hill, A. V., 239, 353 

His, 101 

Hobson, 217 

Hopkins, 5 

Iwanzoff, 178, 201 

Janicki, 92 
Jennings, 7, 357 
Jones, \V. and Folkoff, 31 
Jorgensen, 97 

King, 101 
Koelliker, 76 
Kolzoff, 152 
Komai, 91 
Konopacki, 63, 135 
Korotneff, 91 
Kossel, 5 

Kostanecki, 97, 217 
Kowalewsky, 91, 97 
Kowalewsky et Marion, 91 
Kriiger, 180 
Kiihne, 64 

Lang, 92 

Lavoisier, 4 

Lefevre, 97, 217 

Lewis and Hartmann, 101 

Lewis and Lewis, 63, 79, 81, 335 

Leydig, 57 

Lillie, F. R., 97, 117. J S7^ 163, 

299> 3°7^ 3 2 5, 3 2 6, 334 
Lillie, R. S., 116, 126, 213, 216, 

273. 274 
List, 101 

Loeb, n IT., 216, 219 ff. 
Luckc and McCutcheon, 127 


Ludwig, 96 
Lwoff, 167 
Lynch, 349 

Maas, 91 

MacBride, 203 

Mathews, 61, 212 

McMurrich, 66 

Mead, 164, 167, 216 

Mellor, 25 

Metschnikoff, 87, 89, 91, 92, 117, 177 

Meves, 92, 96, 97, 99, 173, 183, 278 

Minchin, 357 

Montgomery, 364 

Moore, 2 

Morgan, 21, 27, 190, 311, 326 

Packard, 341 
Pasteur, 5 
Patten, 97 
Pereyaslawzewa, 92 
Perez, 317 
Ponder, 119 
Prouho, 93 

Ransom, 101, 117, 286 

Redfield and Bright, 341 

Reed, 320 

Reighard, 189 

Retzius, 213 

Rogers and Cole, 114, 115 

Rohde, 33 

Sauerbruch, 364 

SchafTer, Folkoff and Bayne-Jones, 31 

Scheikewitsch, 66 

Schneider, 178 

Schuberg, 77 

Schumacher, 88 

Scott, 216 

Sedgwick, 33 

Selenka, 92, 93, 96, 277 

Shaerer, 1 13 

Sobotta, 99, 167, 170 

Spallanzani, 188 

Spek, 91 

Spengel, 97 

Storch, 93 



Studnicka, 33 Watase, 267 

Svedberg, 44 Watson, 12 

Weismann, 293 

Tennent, 21, 57 Whitaker, 194 

Theel, 96, 278 Whitman, 12, 33, 122 

Torrey, H. B., 91, 108 Wilson, C. B., 93 

Torrey, J. C, 97 Wilson, E. B., 21, 61, 97, 250, 302, 32: 

Treadwell, 216 Wilson, J. W., 301 

v. Wistinghausen, 97 

Van Beneden, 92 Wohler, 2 

Vejdovsky, 96, 237 Woodger, 34 
Vejdowsky and Mrazek, 96 

Verworn, 2 Yatsu, 91, 93 

Warburg, 113, 118 Zelinka, 93 

Warren, 92 Ziegler, 91, 96, 257, 281 

> \ 

: f * 

« "i 

' - 


Index of Subjects 



Agglutination of spermatozoa, 196 
Alveolar theory of cytoplasmic struc- 
ture, 59-63 
Amia, egg of, 301 
Amino-acids, 39 
Amitosis, 250, 266 

Amoeboid changes in eggs, 89, 93, 108 
Amoeboid movements, 87 

of nerve-cells, 82-85 
Amphibians, ectoplasm of eggs of, 99 
Ampkioxus, ectoplasm of egg of, 99 

egg of, 153, 177,201,266,302 

fertilization in egg of, 167-170 

Anastral mitotic figure, 266 

Jrbacia, egg of, 23, 48, 64, 108, 112, 

116, 117, 126, 127, 173, 187, 

192, 200, 201, 222, 224, 233, 

269, 274, 275, 277, 303, 336 

fertilization in egg of, 170-171 

spermatozoa of, 104 
Arthropods, ectoplasm in eggs of, 98 
Ascaris, egg of, 49, 153, 266 
Asexual reproduction, 319 
Asteracanthioru egg of, 213, 214 
Asterias, egg of, 186, iSS, 201, 212, 

213, 214, 215, 337 
Asters in cell-division, 255 

of first cleavage, 182 
Astral fibres, 69, 215 

rays, 69 
Astropecten, egg of, 215 
Asynchrony of nuclear and cyto- 
plasmic division, 266 
Autolytus varians, eggs of, 57 
Axial gradient, 301 
Axis of embryo, 300 

Bacteria, 31, 32, 35 
ectoplasm in, 88 

Bacterium, 8 

Beroe, egg of, 91 

Bilaterality, 301 

Biogen-molecule, 5 

Biological system, 17 

Biologists, physico-chemical, 14 

Biology, " physico-chemical," 11 

Birds, ectoplasm of eggs of, 101 

Blastomeres, movement of, 335 

Blastula, 301 

Blue-green algae, 31, 32 

Body-fluid, role of, in fertilization, 
196, 198 

Bonellia, egg of, 97 

Brain, development of, 366 

Butyric acid, in experimental partheno- 
genesis, 213, 222 


Cancer, 345~346, 365 
Capillary spaces, 298, 344 
Carbohydrates, 40 

Carbon-dioxide, means for experi- 
mental parthenogenesis, 2 1 2 
Cell, 35 

chemical composition of, 38 ff. 

forms of, 37 

size-variations of, 37 
Cell-division, change in shape of 
echinid egg during, 272 

definition of, 267 

in egg of Arabacia, 255 ff. 

in sea-urchin eggs, 253 ff. 



Cell-division, movements in the cyto- 
plasm during, 267 ff. 

resistance and susceptibility in, 

surface-tension in, 270 IT. 
Cell-lineage, 241, 292 
Cell-membrane, 70-74 
Cells, fixation of, 65-67 

physical properties of, 42 
Cellular oxidation, 349 
Centres of first cleavage spindle, origin 

of, 182 
Centrifuged eggs, 306 
Centriole, 69, 162, 166 
Centrosomes in fertilization, 182, 183 
Centrosphere, 69, 162 

in cell-division, 255 
Cerebratulus, egg of, 93 
Chaeiopterus, egg of, 64, 97, 117, 153, 
177, 191, 223, 270, 297, 299, 

3°3, 317, 34i ? 347 
fertilization in egg of, 164-167 
Chemistry of the cytoplasm, 58 
Chiton, egg of, 97 
Chromatin, increase of, 311 
Chromidia, 56 

Chromosomes, 6, 49, 50, 310 
aberrant behavior of, 35<>-35~ 
degeneration of, 56 
incompatibility of, 351 
individuality of, 321-324 
number of, in parthenogenesis, 210 
role of, in differentiation, 321 ff. 
Ciona, egg of, 195, 201, 266 
Cleavage, classification of, 291 
determinate, 269 
indeterminate, 269 
Cleavage-amphiaster, 167 
Cleavage-asters, 164 

origin of, 182, 183 
Cleavage-centres, 164 
origin of, 182, 183 
Cleavage-cycle, ectoplasmic changes 
during, 281 
maximum susceptibility in, 277 
periods of susceptibility in, 274- 
Cleavage-nuclei, types of, 1S0 
Coelenterata, 300 

Coelenterates, ectoplasm in eggs of, 90 
Colloid, 43, 44 
Colloid chemistry, 59 
Conduction, 358 

in living cells, 119-121 
Contractility in eggs, 116, 117 
Contraction, 358 

in living cells, 123 
Copulation, 155 

in Platynereis, 189 
Corymorpha, egg of, 283 
Crepidula, egg of, 283 
Criteria for normality, 22, 23 
Cross-fertilization, 21, 57, 19S 
Cryptobranchus, egg of, 180 
Crystals in eggs, 57 
Cumingia, egg of, 98, 153, 223 
Cyclops, egg of, 49, 180 
Cynthia, egg of, 304 
Cytolysis in eggs, 228 
Cytolytic agents, 227, 232 
Cytoplasm, 8 

chemistry of, 58 

ground-substance, 57 

movements in, 43, 52, 267-270, 
282, 284, 305, 306, 308 

physical properties of, 58 

role of, in heredity and differen- 
tiation, 326-330 
Cytoplasmic inclusions, 8, 52, 53 ff., 
role of, in differentiation, 302 ff. 
Cytoplasmic reactions, 326 
Cytoplasmic structure, alveolar theory 
of, 59-63 

filar theory of, 63 

granular theory of, 63-64 


Dehydration, 125, 244, 245 

Delamination, 301 

Dentalium, egg of, 97 

Description, role of, in biology, 10, 25 

Differentiation, in cells, 247 

ecto-endoplasmic, 8, 47-49, 363 

nucleo-cytoplasmic, 7, 47 

surface-interior, 357 

without cleavage, 216, 217, 218, 



Dilute sea-water, unfertilized eggs in, 

Dinophilus, egg of, 187 
Drosophila, 321, 328 

Echinarachnius, egg of, 57, 69, 106, 
108, 109, 115, 116, 126, 173, 
181, 187, 192, 199. 201, 222, 

254, 273 
fertilization in egg of, 171-177 
Echinocardium, egg of, 192 
Echinoderms, ectoplasm in eggs of, 


Echinus, eggs of, 106, 192 
Ectoderm, 122, 288, 366 
Ectoplasm, 8 

defined, 75 

in bacteria, 88 

in cells in tissue-culture, S3-S6 

in cells of multicellular organisms, 


in eggs, 89 ff. 

of amphibians, 101 

of Amphioxus, 99 

of arthropods, 98 

of birds, 101 

of Cerebratulus, 93 

of Coelenterata, 90 

of echinoderms, 93-96 

of flatworms, 92 

of insects, 98 

of lamprey, 100 

of Lingula, 93 

of mammals, 101 

of nematodes, 92 

of Phallusia, 98 

of reptiles, 101 

of Rotifera, 93 

of segmented worms, 96 

of selachians, 100 

of sponge, 89 

of teleosts, 101 
in leucocytes, 86 
in muscle-cells, 78-81 
in nerve-cells, 81-85 
in plant-cells, 88 
in protozoa, 87, 88 

Ectoplasm, in red blood cells, 86 
in sponge cells, 75 
increase of during cleavage, 33 1— 

33 2 

necessity of, for fertilization, 

philosophical implications of 
theory of, 365-366 

relation of, to cell, 9 

relation of, to environment, 9 

role of, in differentiation, 332-339 
Ectoplasmic changes due to sperm- 
attachment, 104 
Ectoplasmic structure of eggs revealed 

experimentally, 101-102 
Egg, mammalian, 302 
Egg-cell, 36 

Egg-fragments, development of, 53, 
180, 297, 307 

diploid, 318 
Egg-jelly, role of, in fertilization, 19 
Egg-secretions, effect of, in initiating 

development, 20-21 
Eggs, centrifuged, 306 

colors of, 57 

ectoplasm in, 89 ff. 
Electron-supermicroscope, 26 
Endoderm, 288 
Endoplasm, 8, 4S 

definition of, j6 
Endoplasmic buds, 192 
Ensis, egg of, 98 
Entrance-cone, 177, 17S 
Epiboly, 301 
Ethics, 367 
Exoplasm, 75, 88, 89 
Experiments, control of, 22 

in biology, categories of, 16 

on eggs, 17 

on the living system, 16 

on tissues in vitro and in situ, 19 
Extra-ovates, 24, 336, 337 

Fertilizability of egg of Amphioxus, 1 88 
of Arbacia, 187 
of Asterias, 186 
of Fundulus, 189 



Fertilizability of egg of Nereis, 1 86 

of Platynereis, 189 
Fertilizability, seat of, in eggs, 191 
Fertilizable condition of eggs, 186 
Fertilization, as chemical reaction, 203 

BoverPs theory of, 182 

classes of eggs in, 153 

inhibition of, 196 

phases of, 179, 231 

phenomena common to, 177 

specificity in, 197-198 

straight, 21 

superficial cytolysis — corrective 
factory theory of, 23c 
Fertilization-cone, 159 ff., 178 
Fertilizin, 195-197 
Filaments, ectoplasmic, 79, 80, 85, 

102, 105, 145, 277-280 
Filar theory of cytoplasmic structure, 


Fixation, 16 

of cells, 65-67 
Flatworms, ectoplasm in eggs of, 92 

Gastrula, 213, 300 

Gastrulation, 193, 301 

Gene, 7 

role of, in heredity and differentia- 
tion, 326 

Gene-molecule, S~7^ 323 

Genes, linear arrangement of, 322 

Gene-theory of heredity, 7, 325 

Genetic restriction, 313, 320 

Genetics, 8, 321 

Germ cells, 147, 249, 316 

Germinal vesicle, 131, 153, 316 

Golgi bodies, 55 

Gradient, axial, 301 

Granular theory of cytoplasmic struc- 
ture, 63-64 

Ground-substance, 37, 57-58, 64, 67, 


dehydration of, 245 

in nucleus, 70 

radiating structures in, 68-70 

structure of, 64 
Growth, 248 
Gyrodactylus, egg of, 153 


Haemolytic agents, 227, 232 
Heat-liberation of eggs, 115 
Heat-production in eggs, 114-115 
Hermaphroditism, 155, 156, 329 

in starfish, 213 
Hetero-agglutination of spermatozoa, 

Heteronereis phase, 186 
Hormones, integration by, 122 
Hyaline plasma-layer in sea-urchin 
eggs, 95-96, 107, 171, 257, 
Hydration and dehydration, of yolk- 
spheres, 135 
role of lipoid in, 135 
Hydrogen-ion concentration, effect of 

changes in, on eggs, 340 
Hydrophiliis, egg of, 301 
Hypotonic sea-water, changes of eggs 
in, 130 
development of eggs in, 130 
means for experimental parthe- 
nogenesis, 129 
susceptibility of eggs to, 108- 


Hypotonicity, limits of, 127-129 


Independent irritability, 237 

Indicia, physiological, for normality, 


Individuality of the chromosomes, 

Inhibition of fertilization by body- 
fluid, 198 

Initiation of development in eggs, 206- 

Insects, ectoplasm in eggs of, 98 

Integration of cells, 121-123, 363 

Intercellular connections, 77, 120, 335 
fibres, jj, 78 

Invagination, 193, 301 

Isolated blastomeres, 318 

Kropotkin theory, ^67 



Lamprey, ectoplasm of egg of, 100 

Life-molecule, 5 

Linear arrangement of the genes, 322 

Lingula, ectoplasm in egg of, 93 

Lipins, 39, 53, 54 

Living and non-living, similarities in, 

Living things, limitation of investiga- 
tion, 3 

organization of, 2 

physico-chemical systems, 1 
Loligo, egg of, 301 
Lunar periodicity, 158 
Lysin, 232 


Macira, egg of, 97, 153, 223 

Mammals, ectoplasm of eggs of, 101 

Mathematics, use of in science, 25 

Maturation, 148 

Mayonnaise, 62 

Measurements in biology and physics, 

Mechanistic, use of term in biology, 14 
Median plane, 300 
Medicine, 364 
Medusa-egg, 177 
Membrane, 70-74, 140 

nuclear, 50 

plasma, 72, 140 

precipitation, 73 

semi-permeable, 140 

synaptic, 120 

vitelline, 72, 140 
Membranipora, ectoplasm in egg of, 93 

egg of, 203 
Mendel's laws, 321 
Merogony, 180, 297, 315, 318 
Mesoderm, 288 
Methods of investigation in biology 

and physics, 3 
Micropyle, 201 nx> 
Middle-piece of spermatozoon, 171 ff. 
Migration of cells in tissue-culture, 

Mitochondria, 56 

Mitosis, 250 

Mitotic figure, 59, 240 

Models in biology, 16, 62, 271 

Monaster-stage, 341, 351 

Morula, 301 

Multicellular organism, history of, $6 

Muscle-cells, ectoplasm in, 78-81 

Mutations, 321, 340, 352 

Myiilus, egg of, 97"9 8 

middle-piece of sperm of, 183 
Myzostoma, egg of, 153 


Nematodes, ectoplasm In eggs of, 92 
Nephritis, 139, 365 

Nereis, egg of, 24, 53, 54, 55, 56, 59, 
64, 97, 102, 108, 113, 116, 
129, 134, 135, 138, 139, 153, 
177, 186, 194, 195, 233, 231, 
269, 278, 303, 309, 317, 
34i ? 347 
Nerve-cells, ectoplasm in, 81-85 
Nerve-systems, 358 
Non-mechanistic, 14 
Normal egg, 177 
Normal living organism, 17 
Nuclei, size of, 49 
Nucleic acid in bacteria, 31 
Nuclein, 312 
Nucleolus, 50 
Nucleo-protein, 41, 51 
Nucleus, 8, 31, 51 

chemistry of, 51 

form of, 49 

ground-substance in, 70 

origin of, 357 

resting, 51 

role of, in differentiation, 308 ff. 
Nutrition, 139, 358 

animal and plant, 360 

Oedema, 139, 365 

Oil, function of, in cell, 54 

Oniscus, 66 

Opalina, 33 



Organism, relation to environment, 

226, 356 
Organismal conception, n 
Organism-as-a-whole, 33 
Organization, 14 

of living matter, 2, 7 

of protoplasm, 7 
Organizator theory, 239, 290 
Origin of species, 360-361 
Osmotic method for experimental 

parthenogenesis, 219 
Oxidation, cellular, 118-119, 349 
Oxygen-consumption in eggs, 11 3-1 14 

Pancreas, 122 

Paramoecium, 71, 323 
Parechinus, egg of, 173 
Patella, egg of, 21, 97 
Parthenogenesis, experimental, butyr- 
ic acid in, 221 
chromosomes in, 210 
definition of, 240 
effect of hypertonic sea-water 

in, 218 
effect of hypotonic sea-water 

in, 218 
effect of strong hypertonic 

solutions in, 224 
effect of temperature in, 218 
effect of ultra-violet radiation 

in, 218 
in egg of Amphitrite. 216 
of Arbacia, 219 
of Aster ias, 212 ff. 
of Chaetopterus, 216 
of Cumingia, 216 
of frog, 21 1 
of Mactra, 217 
of Nereis, 217 
of Podarke, 216 
of Strongylocentrotus, 219 
of Thalassema, 217 
lysin-corrective factor method 

in, 221 
osmotic method for, 219 
superficial cytolysis theory of, 

Parthenogenesis, natural, chromo- 
somes in, 20S 
facultative, 208 
fixed, 208 

stages of maturation of eggs in, 

Parthenogenetic egg, haploid, 318 

Pedicellina, egg of, 203 

Pediculopsis, egg of, 180 

Perivitelline space, 72, 105, 108, 162, 
203, 277 

Petromyzon, egg of, 100, 169 

Phagocytosis, 86-87, 145, 146 

Phallusia, egg of, 98, 153, 266 

Pkascolosoma, egg of, 96, 153 

Physical properties of the protoplasm, 
42 ff. 

Physical science, 364 

Physico-chemical biology, 11 
means, 15 

Physics and chemistry, in biology, 17 

Physics, concepts of, 12 

Pigment granules, 57, 269 

Plant-cells, ectoplasm in, 88 

Platynereis, egg of, 20, 24, 54, 55, $6< 
69, 72, 102, 139, 191, 195, 
200, 201, 278 
description of egg of, 47 

Pluripotency, 296, 315 

Polar bodies, 148 

nuclei of, in parthenogenesis, 

suppression of, 342 ff. 

Polarity in eggs, 300 

Polyembryony, 297, 317 

Polyenergid, 35 

Polyploidy, 20, 319 

Polyspermy, 199-203 

Polystomum 7 egg of, 153 

Porifera, 300 

Potencies, of the egg, 327 

of the spermatozoon, 327 

Primitive protoplasm, 355 

Propagation of effect of sperm-attach- 
ment in eggs, 1 16 

Prostheceraeus, egg of, 153 

Protein, 39 

Proteins and specificity, 41 

Protoplasmic organization, 9 



Protoplasmic structure, theories of, 59 
Protoplasmic system as life-unit, ff 
Protozoa, n, 36, 358 

ectoplasm in, 87. 88 
Psammechinus, egg of, 57 
Pseudopodia, ectoplasmic, 81, 82-85, 

Sq, 103 


Quantitative biology, 3, 5, 25, 238 


Radiations as experimental means, 348 
Radium, 341 
Red blood cells, 86 

Red blood corpuscles, 36, 54, 71, 119 
Refractive index, 44 
Regeneration, 24S, 320 
Reptiles, ectoplasm in eggs of, 101 
Resistance and susceptibility in cell- 
division, 272 
Respiration, 358, 359 
Resting nucleus, 51 
Rhabditis, egg of, 180, 181, 206 
Rhynchelmis, egg of, 97 
Roentgen-rays, 341, 349 
Rotifera, ectoplasm in eggs of, 93 

Sabellaria, egg of, 102 

Saccocirrus, egg of, 187 

Sagitta, egg of, 153 

Salinity, effect of changes in, 340 

Sciara, egg of, 351 

Sea-urchin, skeleton of larva of, nor- 
mal variation, 21 

Segmented worms, ectoplasm in eggs 
of, 96 

Selachians, ectoplasm in eggs of, 100 

Self-differentiation, 9, 309 

Self-fertilization, 155 

Self-regulation, 9, 104, 238, 364 

Semi-permeability, 139-146 

Sex-differentiation, 329 

Somatic cells, 147 

Specific gravity, 44 

Specificity. 41 

Specificity in fertilization, 197-198, 

Sperm-amphiaster, 167 
Sperm-aster, 163 
Spermatozoa, origin of, 149 

structure of, 151 
Spermatozoon, middle-piece of, 171 ff. 

of Ascaris, 152 

of CerebratuluS) 151 

of Nereis, 1 5 1 
Sperm-centrosome, 163 
Sperm-nucleus, 163 
Sperm-rotation, 162, 172 
Spindle-threads, 69 
Sponge, ectoplasm in egg of, 89 
Starfish, egg of, 154 
Strongylocentrotus, egg of. 106, 192, 

3°4> 3°S 

Sub-normal eggs in experiment, 19 

Surface-cytoplasm, 8 

Surface-tension, 283 

in cell-division, 270 ff. 

Susceptibility during membrane-sepa- 
ration, 109 ff., 282 

Synaptic membrane, J7, 120 

Syncytia, 33 


Teleosts, ectoplasm of eggs of, 101 
Temperature, as experimental means, 
20, 21S 

effect of change in, on eggs, 340 
Thalassemia, egg of, 97, 153, 223 
Thysanosoon, egg of, 153 
Tissue-culture, cause of migration of 
cells in, 85-86 

cells in, 252 

ectoplasm in cells in, 83-86 
Tissues, behavior of, under laboratory 

conditions, 18 
Trematode eggs, 183, 305 
Trophoblast, 302 
Tubifex, egg of, 317 
Tumors, 43, 249, 320 


Ultra-centrifuge, 39 
Ultra-filterable viruses, 32 



Ultra-microscope, 59 
Ultra-microscopic organisms, 356 
Ultra-violet light, 20 

effect of, on egg of Nereis, 

218,341-344, 34&-347 
Unicellular organism, 35, 249 
Union of egg- and sperm-nuclei, modes 

of, 179 ff. 


Viscosity, 44-46 

Vitalism, 14 

Vitelline membrane, 140 


Water, component in vital reactions, 

distribution of, during cleavage, 

in cells, 41 
movement into and out of cells, 


structural part of living system, 

Water-drops, 126, 132 

and protoplasmic structure, 137 
effect of temperature in producing 

them, 136 
formed by ultra-violet light, 136 
hypertonic sea-water and, 136 
in normal eggs, 137 
in nucleus, 134 
rate of formation of, 134 
rhythmical phenomenon, 137 

Water-level in cells, 143, 243-245 

White blood cell, 365 


X-ray spectroscopy, 39 

Yolk, S3, 54 

role of, in development, 54-55 
Yolk-spheres, fusion of, 131 

polyphasic, 55 

Zygote nucleus, 164